Novel targets and compositions for use in decontamination, immunoprophylaxis, and post-exposure therapy against anthrax

ABSTRACT

The present invention relates to the decontamination of anthrax spores, prophylaxis and treatment of anthrax infections and, more particularly, to compounds that act as specific inhibitors of  B. anthracis  germination/outgrowth-associated proteins, methods and means for making such inhibitors and their use as pharmaceuticals and/or vaccines. The invention also relates to the prophylaxis and treatment of anthrax infections and, more particularly, to vaccines and compositions that comprise  B. anthracis  antigens, epitopes, proteins, or nucleic acid molecules, including anthrax protective antigen, anthrax lethal factor, anthrax edema factor and anthrax proteins associated with spore germination and outgrowth, as well as methods and means for making such compositions and their use pharmaceuticals and/or vaccines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application60/486,369 filed Jul. 11, 2003. Reference is also made to the followingjointly-owned applications and patents: U.S. patent application Ser. No.10/346,021 filed Jan. 16, 2003, which is a continuation-in-part of U.S.patent application Ser. No. 10/116,963, filed Apr. 5, 2002, which is acontinuation-in-part of U.S. patent application Ser. No. 10/052,323,filed Jan. 18, 2002, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/563,826, filed May 3, 2000 (issued Feb. 19, 2002as U.S. Pat. No. 6,348,450), which claims priority from U.S. ProvisionalApplication No. 60/132,216, filed May 3, 1999, and is also acontinuation-in-part of U.S. patent application Ser. No. 09/533,149,filed Mar. 23, 2000, which in turn is a continuation of U.S. patentapplication Ser. No. 09/402,527, filed on Aug. 13, 2000. Each of theseabove-referenced applications and each of the documents cited in each ofthese applications (“application cited documents”), and each documentreferenced or cited in the application cited documents, either in thetext or during the prosecution of those applications, as well as allarguments in support of patentability advanced during such prosecution,are hereby incorporated herein by reference. Various documents are alsocited in this text (“application cited documents”). Each of theapplication cited documents, and each document cited or referenced inthe application cited documents, is hereby incorporated herein byreference.

GOVERNMENT SUPPORT

Research carried out in connection with this invention may have beensupported in part by grants from the Office of Naval Research grantN00014-01-1-0945 and the National Institutes of Health Grant1-R43-AI-47558-01A2. The United States government may have certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the fields of bacteriology,immunology, and vaccine technology.

The present invention also relates to Bacillus anthracis proteins asnovel targets and compositions for use in decontamination,immunoprophylaxis, and post-exposure therapy against anthrax. Theinvention relates to the administration of these proteins by any numberof routes including, but not limited to, mucosal, e.g., intranasal,perlingual, buccal, oral, oral cavity, intravenous, intramuscular,transdermal or intracutaneous, when used in immunoprophylaxis orpost-exposure therapy. The invention further relates to methods ofnon-invasive immunization in an animal and/or methods of inducing animmunological, e.g., systemic immune response or a therapeutic, e.g., asystemic therapeutic response, in an animal against anthrax, productstherefrom and uses for the methods and products therefrom. The inventionyet further relates to such methods comprising contacting the nasalmucosa (e.g. via nasal spray) or the skin of the animal with a vector inan amount effective to induce the response, e.g., systemic immuneresponse against anthrax in the animal. Even further, the inventionrelates to such methods wherein the vector comprises and expresses anexogenous nucleic acid molecule encoding an epitope or gene product ofinterest, e.g., an anthrax antigen. Still further, the invention relatesto such methods wherein the response, e.g., systemic immune ortherapeutic response, can be to or from the epitope or gene product.Even further still, the invention relates to such methods wherein thevector is non-replicative.

The invention yet further still relates to such methods wherein thenucleic acid molecule can be exogenous to the vector. The invention alsorelates to such methods wherein the exogenous nucleic acid moleculeencodes one or more of an antigen or portion thereof, e.g., one or moreof an epitope of interest from Bacillus anthracis, e.g., anthraxprotective antigen, anthrax lethal factor or anthrax proteins associatedwith spore germination or outgrowth.

Even further, the invention relates to such methods wherein the immuneresponse can be induced by the vector expressing the nucleic acidmolecule in the vector or in the animal's cells, e.g., cells lining amucosal surface, such as the nasal mucosal surface, epidermal cellsincluding but not limited to keratinocytes, melanocytes, langerhanscells, merkel cells and hair matrix cells. The invention still furtherrelates to such methods wherein the immune response can be againstBacillus anthracis.

Also, the invention relates to compositions used in the methods. Forinstance, the invention relates to a prophylactic vaccine or atherapeutic vaccine or an immunological composition comprising thevector, wherein the vector can be replicative or non-replicative.

The invention additionally relates to such methods and compositionstherefor wherein the animal can be a vertebrate, e.g., a mammal,advantageously a mammal such as a human or a companion or domesticatedor food- or feed-producing or livestock or game or racing or sportanimal, for instance, a cow, a horse, a dog, a cat, a goat, a sheep or apig.

The invention further relates to such methods and compositions thereforwherein the vector can be one or more of a viral, including viral coat,e.g., with some or all viral genes deleted therefrom, bacterial,protozoan, transposon, and retrotransposon, and DNA vector, e.g., arecombinant vector; an adenovirus, such as an adenovirus defective inits E1 and/or E3 and/or E4 region(s) and/or all adenoviral genes.

The invention further relates to such methods and compositions thereforewherein the vector can be non-replicative.

The invention further relates to mucosal, e.g., intranasal, perlingual,buccal, oral, oral cavity, administration of adenovirus defective in itsE1 and/or E3 and/or E4 region(s) and/or all adenoviral genes,advantageously defective in its E1 and E3 regions, e.g., such anadenovirus comprising an exogenous or heterologous nucleic acidmolecule, such as an exogenous or heterologous nucleic acid moleculeencoding an epitope of interest of an anthrax, e.g., one or more anthraxepitiopes of interest and/or one or more anthrax antigens. Such anadministration can be a method to induce an immunological response, suchas a protective immunological response. The adenovirus in this instancecan be a human adenovirus. The adenovirus can be another type ofadenovirus, such as a canine adenovirus.

The invention still further relates to such methods encompassingapplying a delivery device including the vector to the skin of theanimal, as well as such a method further including disposing the vectorin and/or on the delivery device; and, to such delivery devices.

The invention yet further relates to such methods wherein the vector canhave all viral genes deleted therefrom, as well as to such vectors.

The invention yet further relates to such methods wherein the vector canbe non-replicative bacterial vectors expressing Bacillus anthracisantigens, for example, wherein the bacterial vector has been irradiated.

In addition, the invention relates to immunological products generatedby the expression and the expression products, as well as in in vitroand ex vivo uses thereof.

The invention also relates to such methods wherein the Bacillusanthracis protective antigen or fragments thereof are expressed from theviral or bacterial vectors.

The invention further relates to such methods wherein an immunogenic butatoxic Bacillus anthracis lethal factor is expressed from the viral orbacterial vectors.

The invention yet further relates to such methods wherein immunogenicbut atoxic fragments of the Bacillus anthracis immune inhibitor A,Hypothetical protein 1 (Germaxin), Hypothetical proteins 2, 4, and 5,GPR-like spore protease, CIP protease, cysteine synthase A, heat-shockproteins 70 and 60, class 1 heat shock protein, elongation factor G andTs, RNA polymerase, acetate kinase, delta-1-pyrroline-5-carboxylatedehydrogenase, pyruvate dehydrogenase, alkyl hydroperoxide reductase,oxidoreductase, elonase, ATP synthase, fructose-bisphosphate aldolase,triosephosphate isomerase, glyceraldehyde 3-physphate dehydrogenase,sugar ABC transporter, band 7, and alcohol dehydrogenase are expressedfrom the viral or bacterial vectors.

The invention still further relates to such methods wherein targetingthe Bacillus anthracis alanine racemace induces pre-mature sporegermination for immunoprophylaxis and post-exposure therapy againstanthrax, and decontamination of areas covered by anthrax spores.

BACKGROUND

Anthrax is a zoonotic illness that has been recognized for centuries. Inthe 1870s, Robert Koch demonstrated for the first time the bacterialorigin of a specific disease, with his studies on experimental anthrax,and also discovered the spore stage that allows persistence of theorganism in the environment. Shortly afterward, Bacillus anthracis wasrecognized as the cause of woolsorter disease, now known as inhalationalanthrax. The development of vaccines against anthrax began in 1880 withWilliam Greenfield's successful immunization of livestock againstanthrax and Louis Pasteur's 1881 trial of a heat-cured anthrax vaccinein sheep.

Bacillus anthracis is a large, gram-positive, sporulating rod, withsquare or concave ends. Growing readily on sheep blood agar, B.anthracis forms rough, gray-white colonies of four to five micrometer,with characteristic comma-shaped or “comet-tail” protrusions. Severaltests are helpful in differentiating B. anthracis from other Bacillusspecies. Bacillus anthracis is characterized by an absence of thefollowing: hemolysis, motility, growth on phenylethyl alcohol bloodagar, gelatin hydrolysis, and salicin fermentation. Bacillus anthracismay also be identified by the API-20E and API-50CHB systems used inconjunction with the previously mentioned biochemical tests. Definitiveidentification is based on immunological demonstration of the productionof protein toxin components and the poly-D-glutamic acid capsule,susceptibility to a specific bacteriophage, and virulence for mice andguinea pigs.

Naturally occurring human cases of anthrax are invariably zoonotic inorigin, with no convincing data to suggest that human-to-humantransmission has ever taken place. Primary disease takes one of threeforms: (1) cutaneous, the most common, which results from contact withan infected animal or animal product; (2) inhalational, which is muchless common and results from spore deposition in the lungs; and (3)gastrointestinal, which is due to ingestion of infected meat. Mostliterature cites cutaneous disease as constituting the large majority(up to 95%) of naturally occurring exposure cases.

Anthrax has been studied for use as a biological weapon for over 80years, with the transmission of spores through air as the most likelymethod of transmission, resulting in inhalational anthrax. Due to therapidly fatal hemorrhagic mediastinitis caused by inhalation of anthraxspores, the dissemination of airborne spores in a populated area couldbe devastating.

Disease occurs when spores enter the body, germinate to the bacillaryform, and multiply. In cutaneous disease, spores gain entry throughcuts, abrasions, or in some cases through certain species of bitingflies. Germination is thought to take place in macrophages, and toxinrelease results in edema and tissue necrosis but little or no purulence,probably because of inhibitory effects of the toxins on leukocytes.Generally, cutaneous disease remains localized, although if untreated itmay become systemic in up to 20% of cases, with dissemination via thelymphatics. In the gastrointestinal form, B. anthracis is ingested inspore-contaminated meat, and may invade anywhere in the gastrointestinaltract. Transport to mesenteric or other regional lymph nodes andreplication occur, resulting in dissemination, bacteremia, and a highmortality rate. As in other forms of anthrax, involved nodes show animpressive degree of hemorrhage and necrosis.

The pathogenesis of inhalational anthrax is more fully studied andunderstood than that of cutaneous or gastrointestinal anthrax. Inhaledspores are ingested by pulmonary macrophages and carried to hilar andmediastinal lymph nodes, where they germinate and multiply, elaboratingtoxins and overwhelming the clearance ability of the regional nodes.Bacteremia occurs, and death soon follows.

Penicillin remains the drug of choice for treatment of susceptiblestrains of anthrax, with ciprofloxacin and doxycycline employed assuitable alternatives. Some data in experimental models of infectionsuggest that the addition of streptomycin to penicillin may also behelpful. Penicillin resistance remains extremely rare in naturallyoccurring strains; however, the possibility of resistance should besuspected in a biological warfare attack. Since reports in 1999 that ananthrax strain had been engineered to be resistant to the tetracyclineand penicillin classes of antibiotics, ciprofloxacin is the recommendedtreatment for adults with suspected inhalational anthrax. The moresevere forms require intensive supportive care and have a high mortalityrate despite optimal therapy.

The virulence of B anthracis is mediated by two plasmids, pXO1 and pXO2,which encodes genese involved in toxin production and capsule formation,respectively. The pXO1 genes pagA, lef, and cya encode the tripartitetoxin protective antigen (PA)-lethal factor (LF)-edema factor (EF)associated with B. anthracis pathogenicity (Inglesby 2002). Productionof PA-LF-EF peaks during the late exponential phase of vegetative growth(Liu 2004). The importance of a toxin in pathogenesis was demonstratedin the early 1950s, when sterile plasma from anthrax-infected guineapigs caused disease when injected into other animals (Smith and Keppie1954). It has since been shown that the anthrax toxins are composed ofthree entities, which in concert lead to some of the clinical effects ofanthrax (Stanley and Smith 1961; Beall 1962). The first of these,protective antigen (PA), is an 83 kd protein so named because it is themain protective constituent of anthrax vaccines. Protective antigenbinds to a cellular receptor (Bradley 2001) and is proteolyticallycleaved by cell surface furin to produce a 63-KD fragment (PA63). Asecond binding domain is then exposed on the 63 kd remnant, whichcombines with either edema factor (EF), an 89 kd protein, to form edematoxin, or lethal factor (LF), a 90 kd protein, to form lethal toxin(Leppla 1990). This occurs through the receptor-bound PA63, whicholigomerizes to a heptamer and acts to translocate the catalyticmoieties of the toxin, LF and/or EF, from endosomes to the cytosol(Singh 1999). Edema factor, a calmodulin-dependent adenylate cyclase,acts by converting adenosine triphosphate to cyclic adenosinemonophosphate. Intracellular cyclic adenosine monophosphate levels arethereby increased and neutrophil functions are impaired, leading to theedema characteristic of the disease (Leppla 1982; Swartz 2001; Inglesby2002). Lethal factor appears to be a zinc metalloprotease and has beendemonstrated to lyse macrophages at high concentration (Bradley 2001),while inducing the release of tumor necrosis factor α and interleukin 1βat lower concentrations (Friedlander 1986; Hanna 1993), which have beenlinked to the sudden death in anthrax infection (Swartz 2001; Inglesby2002).

Although anthrax vaccination dates to the early studies of Greenfieldand Pasteur, the “modern” era of anthrax vaccine development began witha toxin-producing, unencapsulated (attenuated) strain in the 1930s.Administered to livestock as a single dose with a yearly booster, thevaccine was highly immunogenic and well tolerated in most species,although somewhat virulent in goats and llamas. This preparation isessentially the same as that administered to livestock around the worldtoday. The first human vaccine was developed in the 1940s fromnonencapsulated strains. This live spore vaccine, similar to Sterne'sproduct, is administered by scarification with a yearly booster. Studiesshow a reduced risk of 5- to 15-fold in occupationally exposed workers(Shlyakhov and Rubinstein 1994).

To date, there have been many attempts to improve the safety profile andimmunogenicity of anthrax vaccines by using PA as an antigen. Theseattempts include the formulation of PA in adjuvants (Ivins 1992), theuse of purified PA (Singh 1998), the development of PA-based DNAvaccines (Gu 1999), and the expression of PA in Salmonella typhimurium(Coulson 1994).

British and U.S. vaccines were developed in the 1950s and early 1960s,with the U.S. producing an aluminum hydroxide-adsorbed, cell-freeculture filtrate of an unencapsulated strain (V770-NP 1-R) containing PAas the major protective immunogen, and the British vaccine analum-precipitated, cell-free filtrate of a Sterne strain culture. TheU.S. vaccine has been shown to induce high levels of antibody only toprotective antigen, while the British vaccine induces lower levels ofantibody to protective antigen but measurable antibodies against lethalfactor and edema factor (Turnbull 1986; Turnbull 1988). Neither vaccinehas been examined in a human clinical efficacy trial (Inglesby 2002). Ahigh number of the recipients of the vaccine have reported some type ofreaction to vaccination although most were minor. Manufacturer labelingfor the current Michigan Department of Public Health anthrax vaccineadsorbed (AVA) product cites a 30% rate of mild local reactions and a 4%rate of moderate local reactions with a second dose. The current complexdosing schedule for the AVA vaccine consists of 0.5 mL administeredsubcutaneously at 0, 2, and 4 weeks, and 6, 12, and 18 months, followedby yearly boosters. Animal studies examining the efficacy of availableanthrax vaccines against aerosolized exposure have been performed. Whilesome guinea pig studies question vaccine efficacy, primate studies havesupported its role. In recent work, rhesus monkeys immunized with 2doses of the AVA vaccine were challenged with lethal doses ofaerosolized B anthracis spores. All monkeys in the control group died 3to 5 days after exposure, while the vaccinated monkeys were protected upto 2 years after immunization (Ivins 1996). Another trial used the AVAvaccine in a 2-dose series with a slightly different dosing interval,and again found it to be protective in all rhesus monkeys exposed tolethal aerosol challenge (Pitt 1996). Thus, available evidence suggeststhat two doses of the current AVA vaccine should be efficacious againstan aerosol exposure to anthrax spores. However, one significantlimitation on the use of vaccines is that existing vaccines provide noprotection against a number of strains of B. anthracis. Additionally,the current requirement for multiple injections resulting local pain andedema suggests an effective alternative is needed (Joellenbeck 2002).The Georgian/Russian anthrax vaccine consists of live spores from aSterne strain of B. anthracis that is administered in the shoulder byscarification. This vaccine also has several undesirable side effectsand unknown efficacy (Demicheli 1998). Other alternatives that have beeninvestigated include a highly purified, minimally reactogenic,recombinant protective antigen vaccine using aluminum as well as otheradjuvants, cloning the protective antigen gene into a variety ofbacteria and viruses, and the development of mutant, avirulent strainsof B. anthracis.

Further underscoring the need for a new class of anthrax vaccines orremedies is the development of enabling technology capable ofengineering B. anthracis spores into vectors expressing unpredictabletoxins by replacing pXO1 with an artificial plasmid, owing to thedispensability of the pXO1 plasmid for growth of B. anthracis (Welkos2001).

In the 2001 U.S. anthrax attacks, anthrax spores were enclosed inletters and envelopes sent through the mail and resulted in bothcutaneous anthrax (11 cases: 7 confirmed, 4 suspected) in those whohandled such letters, and inhalational anthrax (11 cases) (Centers forDisease Control and Prevention 2001). Other known experiences withlarge-scale, non-naturally occurring anthrax exposure are limited to the1979 accidental release of anthrax spores from a bioweapons factory inSverdlovsk, Russia.

These recent incidents, which also include the suspected use ofbiological and chemical weapons during the Persian Gulf War, underscorethe threat of biological warfare either on the battlefield or byterrorists. Anthrax has been the focus of much attention as a potentialbiological warfare agent for at least six decades, and modeling studieshave shown the potential for use in an offensive capacity. Dispersalexperiments with the simulant Bacillus globigii in the New York subwaysystem in the 1960s suggested that release of a similar amount of B.anthracis during rush hour would result in 10,000 deaths. On a largerscale, the World Health Organization estimated that 50 kg of B.anthracis released upwind of a population center of 500,000 would resultin up to 95,000 fatalities, with an additional 125,000 personsincapacitated (Huxsoll 1989). Both on the battlefield and in a terroriststrike, B. anthracis has the attribute of being potentially undetectableuntil large numbers of seriously ill individuals present withcharacteristic signs and symptoms of inhalational anthrax. Given thesefindings, efforts to prevent the disease or to ameliorate or treat itseffects are of obvious importance. The U.S. military's current M17 andM40 gas masks provide excellent protection against the 1 to 5 μmparticulates needed for a successful aerosol attack. Assuming a correctfit, these masks would be highly effective if in use at the time ofexposure. Some protection might also be afforded by various forms ofshelter.

Until recently, the AVA anthrax vaccine was supplied only to theDepartment of Defense for vaccination of soldiers. This use has recentlyexpanded to include vaccination of reporters who would face potentialexposure while covering any future warfare or terrorist situations. Atthis time, vaccination of the general public is not encouraged by theCenters for Disease Control.

Due to the limited use of the available anthrax vaccines and theirlimited ability to prevent infection caused by numerous anthrax strains,it is therefore apparent that while certain prophylactic and treatmentschemes may prove useful in preventing or ameliorating anthraxinfections, there remains a compelling need to improve the arsenal oftechniques and agents available for this purpose.

Contemporary anti-anthrax remedies focus on the three-component toxinsystem protective antigen (PA)-lethal factor (LF)-edema factor (EF) thatis produced during multiplication of the vegetative form of B. anthracisin the host (Mock and Fouet 2001). The dissemination of an odorless andinvisible aerosol containing PA-free anthrax spores encoding exogenoustoxins would be devastating, as all PA-targeted anthrax vaccines (Price2001; Welkos 2001; Joellenbeck 2002; Rhie 2003; Tan 2003) and remedies(Sellman 2001) are ineffective in protection against anthrax strainswithout PA. Furthermore, although targeting PA has proven effective tovarying degrees of success in counteracting anthrax, it is unknown atthis time whether any of the PA-targeted methods can protect humansagainst inhalational anthrax (Inglesby 2002) during a massive onslaughtwith airborne anthrax spores used as a bioweapon.

A solution to this dilemma might be found at the level of sporegermination and early outgrowth.

The present invention identifies B. anthracis spore proteins as noveltargets in decontamination, immunoprophylaxis, and post-exposure therapyagainst anthrax.

During the life cycle of B. anthracis, the germinating spores are likelythe weakest link in the cycle, akin to plant seedlings and animalbabies, and hence are the easiest vaccine target. Additionally,germination is an upstream event during the life cycle of B. anthracis;arrest of spore germination will preclude any downstream eventsincluding the production of PA-LF-EF. Consequently, blocking sporegermination and early outgrowth provides a logical means to arrestbioengineered anthrax spores regardless of the exogenous toxin they mayencode.

Additionally as previously mentioned, PA, LF, and EF are encoded by aplasmid in B. anthracis. Technology that enables the replacing of thisplasmid with others encoding other unpredictable toxins (e.g., tetanustoxin, cobratoxin, etc.) is not beyond reach. Bioterrorists may bypassany PA-targeted remedies by launching an attack with anthrax sporesproducing toxins other than PA-LF-EF. Therapies that target thegerminating spores would prevent the production of such toxins fromoccurring. Arrest of spore germination with vaccines and/or inhibitorsoverall may prevent utilization of anthrax spores as bioweapons.

Furthermore, factors associated with spore germination or outgrowth maybe highly conserved and can hardly be altered, making these proteins anideal target that should be conserved between strains. In contrast,vegetative cells undergo mutations over time. Development of antibioticresistance in vegetative cells is one such example.

The present invention also provides a new composition for vaccinationagainst Bacillus anthracis, using adenovirus and bacterium vectorednasal and epicutaneous vaccines that can provide protection to largenumbers of people in a timely manner by non-medical personnel.

There are several noteworthy reasons for utilizing recombinant Ad vectoras a vaccine carrier. These include (i) Ad vectors are capable oftransducing both mitotic and postmitotic cells in situ (Shi 1999), (ii)stocks containing high titers of virus (greater than 10¹¹ pfu[plaque-forming units] per ml) can be prepared, making it possible totransduce cells in situ at high multiplicity of infection (MOI), (iii)the vector is safe based on its long-term use as a vaccine, (iv) thevirus is capable of inducing high levels of transgene expression (atleast as an initial burst), and (v) the vector can be engineered to agreat extent with versatility. Recombinant Ad vectors have been utilizedas vaccine carriers by intranasal, epicutaneous, intratracheal,intraperitoneal, intravenous, subcutaneous, and intramuscular routes.

Ad-vectored nasal vaccine appears to be more effective in eliciting animmune response than injection of DNA or topical application of Ad (Shi2001). Previously reported results have shown that the potency of theE1/E3-defective Ad5 vector as a nasal vaccine carrier is not suppressedby any preexisting immunity to Ad (Xiang 1996; Shi 2001).

Furthermore, it is possible to create an epicutaneous vaccine usingrecombinant Escherichia coli vectors as the carrier. Expression ofheterologous genes in recombinant E. coli vectors about two decades ago(Itakura 1977; Goeddel 1979; Goeddel 1979) allowed E. coli cells to beutilized as protein factories for production of exogenous proteinsincluding a variety of vaccines. It was subsequently demonstrated thatrecombinant plasmid DNA extracted from E. coli vectors could beinoculated into animals to elicit an immune response against antigensencoded by the plasmid, the so called genetic immunization or DNA-basedvaccination (Tang 1992; Ulmer 1993). Both approaches required thedisruption of E. coli cells prior to inoculation into animals, inconjunction with subsequent extraction and purification of recombinantprotein and DNA, respectively; it is hazardous to inject undisrupted E.coli cells into humans as a vaccine due to the presence of endotoxin. Ithas recently been demonstrated that topical application of live orirradiated E. coli cells may be a more potent vaccination modality thaninjection of DNA. It is also believed that the skin is able to disruptE. coli cells following topical application and the present inventionhypothesizes that the antigen may be captured from disrupted E. colicells in the outer layer of skin followed by antigen presentation andthe elicitation of protective immunity against pathogens, includingBacillus anthracis. Topical application of E. coli cells as avaccination modality does not pose a biosafety concern because the skinis already in frequent contact with E. coli cells in the environment.Moreover, the biosafety margin of this modality can be further amplifiedby making recombinant E. coli vectors replication incompetent, forexample, with γ-irradiation.

OBJECT AND SUMMARY OF THE INVENTION

The present invention relates to the prophylaxis and treatment ofanthrax infections and decontamination of anthrax spores, moreparticularly, to compounds that act as specific inhibitors or enhancersfor B. anthracis spore germination, methods and means for making suchinhibitors and enhancers and their use as pharmaceuticals, vaccinesand/or decontamination agents.

Accordingly, it is an object of the invention described herein toprovide compositions that are capable of precisely targeting B.anthracis spore proteins without producing significant undesirable sideeffects.

Another object of the invention relates to the prophylaxis and treatmentof anthrax infections and, more particularly, to vaccines andcompositions that comprise B. anthracis antigens, epitopes, proteins, ornucleic acid molecules, including anthrax protective antigen, anthraxlethal factor, anthrax edema factor and anthrax germination/outgrowthassociated proteins, as well as methods and means for making suchcompositions and their use as pharmaceuticals, vaccines, and/ordecontamination agents.

Additionally, it is an object of the invention that viral or bacterialvectors such as Escherichia coli or adenovirus may be useful in makingthe compositions of the present invention and in expressing the B.anthracis antigens, epitopes, proteins or nucleic acid molecules of thepresent invention.

Both non-invasive vaccination onto the skin (NIVS) and intranasalapplication can improve vaccination schemes because skin and nasalmucosa are immunocompetent tissues and this non-invasive procedurerequires no specially trained personnel. Skin-targeted non-invasive genedelivery can achieve localized transgene expression in the skin and theelicitation of immune responses (Tang 1997). These results indicate thatvector-based NUVS is a novel and efficient method for the delivery ofvaccines. The simple, effective, economical and painless immunizationprotocol of the present invention should make vaccination less dependentupon medical resources and, therefore, achieve vaccination of largenumbers of individuals against anthrax in a timely manner by non-medicalpersonnel.

Accordingly, an object of the invention can be any one or more of:providing a method for inducing an immunological response, e.g.,protective immunological response, and/or a therapeutic response in ahost or animal, e.g., vertebrate such as mammal, comprising nasally ortopically administering a vector that comprises and expresses a nucleicacid molecule encoding a gene product that induces or stimulates theresponse; such a method wherein the nucleic acid molecule isheterologous and/or exogenous with respect to the host; mucosal, e.g.,intranasal, perlingual, buccal, oral, oral cavity administration ofadenovirus defective in its E1 and/or E3 and/or E4 region(s) and/or alladenoviral genes, advantageously defective in its E1 and E3 and E4regions, e.g., such an adenovirus comprising an exogenous orheterologous nucleic acid molecule, such as an exogenous or heterologousnucleic acid molecule encoding an epitope of interest of anthrax, e.g.,one or more Bacillus anthracis epitiopes of interest and/or one or moreBacillus anthracis antigens; such an administration wherein animmunological response, such as a protective immunological response isinduced; products for performing such methods; uses for such methods andproducts, inter alia.

The present invention provides a method of non-invasive immunization inan animal, comprising the step of: contacting skin or nasal, oral,perlingual or buccal mucosa of the animal with a vector in an amounteffective to induce an immune response in the animal. The invention alsoprovides a method for immunizing animals comprising the step ofskin-targeted non-invasive delivery of a preparation comprising vectors,whereby the vector is taken up by epidermal cells and has an immunogeniceffect on vertebrates. The invention further provides a method forimmunizing animals by a delivery device, comprising the steps ofincluding vectors in the delivery device and contacting the naked skinof a vertebrate with a uniform dose of vaccines confined within thedevice, whereby the vector is taken up by epidermal cells for expressingand/or presenting a specific antigen in the immunocompetent skin tissue.The invention further provides a method for immunizing animals by adelivery device, comprising the steps of including vectors in thedelivery device and contacting the nasal mucosa of a vertebrate with auniform dose of vaccines confined within the device, whereby the vectoris taken up by the mucosa for expressing and/or presenting a specificantigen in the immunocompetent tissue. The vector may be adenovirusrecombinants, DNA/adenovirus complexes, DNA/liposome complexes,bacterial vectors containing recombinant plasmids, or other vectorscapable of expressing antigens in the skin or mucosa of a vertebrate.

In a preferred embodiment of the present invention, the vector isnon-replicative. For example, the vector can be irradiated.

In an embodiment of the present invention, there is provided a method ofinducing an immune response, comprising the step of: contacting skin, oranal, oral perlingual or buccal mucosa of an individual or animal inneed of such treatment by topically applying to said skin animmunologically effective concentration of a recombinant vector encodinga gene of interest.

In another embodiment of the present invention, there is provided amethod of inducing a protective immune response in an individual oranimal in need of such treatment, comprising the step of: contacting theskin or nasal mucosa of said animal by topically applying to said skinan immunologically effective concentration of a vector encoding a genewhich encodes an antigen which induces a protective immune effect insaid individual or animal following administration. In yet a furtherembodiment of the invention, contacting the skin may include the use ofmicroneedles or similar devices. For example, “Microfabricatedmicroneedles: a novel approach to transdermal drug delivery” describesthe benefits associated with the use of microneedles, and would allowone of skill in the art to practice the present invention in combinationwith such devices {Henry, 1998 #134}. In certain instances, the use ofsuch devices may be considered “non-invasive.”

The invention thus provides methods of non-invasive immunization in ananimal and/or methods of inducing an immune, e.g., systemic immune, ortherapeutic response in an animal, products therefrom and uses for themethods and products therefrom. The invention further provides suchmethods comprising contacting skin or nasal mucosa of the animal with avector in an amount effective to induce the response, e.g., immuneresponse such as systemic immune response or therapeutic response, inthe animal. Even further, the invention provides such methods whereinthe vector comprises and expresses an exogenous nucleic acid moleculeencoding an epitope or gene product of interest. Still further, theinvention provides such methods wherein the systemic immune response canbe to or from the epitope or gene product.

The invention additionally provides such methods wherein the nucleicacid molecule can be exogenous to the vector. The invention alsoprovides such methods wherein the exogenous nucleic acid moleculeencodes one or more of an antigen of interest or portion thereof, e.g.,an epitope of interest, from a pathogen; for instance, one or more ofanthrax protective antigen, anthrax lethal factor, anthrax edema factor,or Bacillus anthracis proteins associated with germianation/outgrowth;and/or a therapeutic and/or an immunomodulatory gene, such as aco-stimulatory gene, a chemokine gene and/or a cytokine gene. See alsoU.S. Pat. No. 5,990,091, WO 99/60164 and WO 98/00166 and documents citedtherein.

Even further, the invention provides such methods wherein the immuneresponse can be induced by the vector expressing the nucleic acidmolecule in the vector and/or in the animal's cells, e.g., epidermalcells. The invention still further provides such methods wherein theimmune response can be against a pathogen.

Also, the invention provides compositions used in the methods. Forinstance, the invention provides a prophylactic vaccine or a therapeuticvaccine or an immunological or a therapeutic composition comprising thevector, e.g., for use in inducing or stimulating a response via topicalapplication and/or via mucosal and/or nasal and/or perlingual and/orbuccal and/or oral and/or oral cavity administration. The invention alsoprovides compositions comprising a non-replicative vector. Additionally,the invention also provides compositions comprising a vector or anon-replicative vector, in combination with an adjuvant.

The invention additionally provides to such methods and compositionstherefor wherein the animal can be a vertebrate, e.g., a mammal, such ashuman, or a domesticated or companion or feed-producing orfood-producing or livestock or game or racing or sport animal such as acow, a dog, a cat, a goat, a sheep, a horse, or a pig.

The invention further provides such methods and compositions thereforwherein the vector can be one or more of a virus, including viral coat,e.g., with some or all viral genes deleted therefrom, bacterial,protozoan, transposon, retrotransposon, and DNA vector, e.g., arecombinant vector; an adenovirus, such as an adenovirus defective inits E1 and/or E3 and/or E4 region(s) and/or all adenoviral genes. Theinvention further provides such methods and compositions thereforewherein the vector can be chosen from yeast vectors, insect cellstransduced with baculovirus vectors, or tissue culture cells, andwherein the vector is non-replicative. For example, the vector can beirradiated.

The invention further provides such methods and compositions thereforwherein the vector can be an Escherichia bacterial vector. Furtherstill, the invention provides such methods and compositions thereforwherein the vector is preferably an Escherichia coli bacterial vector.

The invention further provides intranasal and/or mucosal and/orperlingual and/or buccal and/or oral and/or oral cavity administrationof adenovirus defective in its E1 and/or E3 and/or E4 region(s) and/orall adenoviral genes, advantageously defective in its E1 and E3 and E4regions, e.g., such an adenovirus comprising an exogenous orheterologous nucleic acid molecule, such as an exogenous or heterologousnucleic acid molecule encoding an epitope of interest of Bacillusanthracis, e.g., one or more of anthrax protective antigen, anthraxlethal factor, anthrax edema factor, or anthrax proteins associated withspore germination/outgrowth. Such an administration can be a method toinduce an immunological response, such as a protective immunologicalresponse. The adenovirus in this instance can be a human adenovirus. Theadenovirus can be another type of adenovirus, such as a canineadenovirus.

The invention still further provides such methods encompassing applyinga delivery device including the vector to the skin of the animal, aswell as such a method further including disposing the vector in and/oron the delivery device; and, to such delivery devices.

The invention yet further provides such methods wherein the vector canhave all viral genes deleted therefrom, as well as to such vectors.

In addition, the invention provides gene products, e.g., expressionproducts, as well as immunological products (e.g., antibodies),generated by the expression, cells from the methods, as well as in invitro and ex vivo uses thereof. The expression products andimmunological products therefrom can be used in assays, diagnostics, andthe like; and, cells that express the immunological products and/or theexpression products can be isolated from the host, expanded in vitro andre-introduced into the host.

Even further still, while non-invasive delivery is desirable in allinstances of administration, the invention can be used in conjunctionwith invasive deliveries; and, the invention can generally be used aspart of a prime-boost regimen. For instance, the methods of the presentinvention can be used as part of a prime-boost regimen wherein vaccinesare administered prior to or after or concurrently with anotheradministration such as a non-invasive or an invasive administration ofthe same or a different immunological or therapeutic ingredient, e.g.,before, during or after prime vaccination, there is administration byinjection or by non-invasive methods described in this invention of adifferent vaccine or immunological composition for the same or similarpathogen such as a whole or subunit vaccine or immunological compositionfor the same or similar pathogen whose antigen or epitope of interest isexpressed by the vector in the non-invasive administration.

The present invention further comprises the use of the topicalapplication of recombinant vectors as previously described for use inthe administration of genes encoding antigens of interest, expressionproducts, or immunological products, all of which can be used to inducea therapeutic effect.

The present invention also encompasses delivery devices (bandages,adhesive dressings, spot-on formulation and its application devices,pour-on formulation and its application devices, roll-on formulation andits application devices, shampoo formulation and its application devicesor the like) for the delivery of skin-targeted and other non-invasivevaccines or immunological compositions and uses thereof, as well ascompositions for the non-invasive delivery of vectors; and, kits for thepreparation of compositions for the non-invasive delivery of vectors.Such a kit comprises the vector and a pharmaceutically acceptable orsuitable carrier or diluent and an optional delivery device, each in itsown packaging; the packaging may be included in a unitary container orthe packaging may each be in separate containers or each may be in itsown separate container; the kit can optionally include instructions foradmixture of the ingredients and/or administration of the composition.

Pour-on and spot-on formulations are described in U.S. Pat. Nos.6,010,710 and 5,475,005. A roll-on device is also described in U.S. Pat.No. 5,897,267. The contents of U.S. Pat. Nos. 6,010,710, 5,475,005 and5,897,267 are hereby incorporated herein by reference, together withdocuments cited or referenced therein and all documents cited orreferenced in such documents. Moreover, a skilled artisan also knows howto make shampoo formulation as well as devices to apply the formulationto an animal.

It is noted that in this disclosure, terms such as “comprises”,“comprised”, “comprising” and the like can have the meaning attributedto it in U.S. patent law; e.g., they can mean “includes”, “included”,“including” and the like.

These and other objects, features, and advantages of the inventionbecome further apparent in the following detailed description of theinvention when taken in conjunction with the accompanying drawings whichillustrate, by way of example, the principles of this invention.

BRIEF DESCRIPTION OF THE DRAWING

The following Detailed Description, given to describe the invention byway of example, but not intended to limit the invention to specificembodiments described, may be understood in conjunction with theaccompanying Figures, incorporated herein by reference, in which:

FIG. 1. B. anthracis growth

(1A) Growth curve of B. anthracis. (1B) Spore germination and outgrowthat the onset of the B. anthracis' growth curve. Growth was measured byperiodically reading the optical density of culture at 560 nm afterinoculating 1.6×10⁹ colony-forming units (cfu) of B. anthracis dormantspores into 10 ml of the modified nutrient broth medium, followed byincubation at 37° C. with shaking. Data represent one of threeindependent experiments.

FIG. 2. Two-dimensional proteomic profiles of Bacillus anthracisgerminating spores.

2-D proteomic profiles were obtained by subjecting spore proteinscollected at 0 (2A) and 10 (2B) min after incubation to IEF in lineargradient Immobiline Dry-Strips of pH 4-7, separation according to massvia SDS-PAGE, and silver-staining. 2C depicts a bar graph showing thelevels of proteins which were quantified using PDQuest software.Differences were expressed as a percent of the control (dormant sporeprotein). Statistical analysis of data from seven independentexperiments was performed using Student's t test. Differences from thecontrol group reaching statistical significance were indicated by asingle asterisk (p<0.05) or double asterisk (p<0.01). Error bars denotemean±SD.

FIG. 3. Identification of germination/outgrowth-associated proteins ofBacillus anthracis by mass spectrometry.

Fingerprint mass spectra and the predicted peptide fragmentscorresponding to observed m/z values for (3A) spot 12; (3B) spot 13;(3C) spot 19; and (3D) spot 15 are shown. The tryptic autodigestive peakat m/z value 2164.04 (asterisk) served as an internal calibrationstandard.

FIG. 4. Q-TOF 2 MS/MS peptide sequencing.

4A is a diagram showing designations for fragment ions from a peptide.4B is an internal peptide of spot 12 with m/z value at 1465 analyzed byMALDI-TOF MS (FIG. 3A) and was sequenced by Q-TOF 2 MS/MS asGIGLATYLDQSVTK. 4C is an internal peptide of spot 13 with m/z value at1721 from MALDI-TOF MS fingerprint (FIG. 3B) and was sequenced asQSEPVYETTLADLQK.

FIG. 5. Protection of mice against B. anthracis Sterne spores.

FIGS. 5A-5D depict AJ and ICR mice immunized by intranasal and topicalapplication of Ad-vectored anthrax vaccines. FIG. 5A depicts groups A,B, C, D, and E, each of which received topical application of 10⁸ pfu ofAd-LF7, topical application of 10⁷ pfu of Ad-LF7, topical application of10⁶ pfu of Ad-LF7, topical application of 10⁸ pfu of AdCMV-tetC, andintranasal instillation of 10⁷ pfu of Ad-LF7, respectively.

FIG. 5B depicts groups F, G, and H, each of which received topicalapplication of 10⁸ pfu of Ad-PA63, topical application of 10⁸ pfU ofAd-LF7 and 10⁸ pfu of Ad-PA63, and intranasal instillation of 10⁷ pfu ofAd-PA63, respectively.

FIG. 5C depicts groups I, J, K, and L, each of which received topicalapplication of 10⁸ pfu of Ad-LF7, topical application of 10⁷ pfu ofAd-LF7, topical application of 10⁸ pfu of AdCMV-tetC, and intranasalinstillation of 107 pfu of Ad-LF7, respectively.

FIG. 5D depicts groups M, N, and O, each of which received topicalapplication of 10⁸ pfu of Ad-PA63, topical application of 10⁸ pfu ofAd-LF7 and 10⁸ pfu of Ad-PA63, and intranasal instillation of 10⁷ pfuofrAd-PA63, respectively.

FIG. 5E depicts four groups who each received intranasal application ofAdtetC, AdPA83, AdLF7 or AdPA83 and AdLF7, respectively.

FIG. 6. ELISA antibody titers in mice following intranasal and topicalapplication of Ad-vectored anthrax vaccines.

FIG. 6A-D depict bar graphs showing the Elisa titers for the groupsimmunized above in FIG. 5. In each graph, the open column is seraharvested one month postimmunization; stippled column is sera harvestedtwo months postimmunization; and the solid column is sera harvestedeight months postimmunization.

FIG. 6A includes groups A, G, and E. FIG. 6B includes groups F, G, andH. FIG. 6C includes groups I, N and L. FIG. 6D includes groups M, N, andO. Shown are geometric means.

FIG. 7. Toxin-neutralization antibody titers in mice followingintranasal and topical application of Ad-vectored anthrax vaccines.

FIG. 7 shows graphs of the percent cell viability of those groupsimmunized as described in FIG. 5. FIG. 7A includes groups A, E, G, F,and H. FIG. 7B includes groups I, L, M, N, and O. FIG. 7C depicts fourgroups who each received intranasal application of AdtetC, AdPA83, AdLF7or AdPA83 and AdLF7, respectively.

FIG. 8. ELISA titers of anti-LF antibodies generated by topicalapplication of E. coli-vectored vaccines onto mice.

FIG. 8 is a bar graph showing ELISA titers from: Ec-LF4, E. coli cellsharboring pnirB-LF4 without boost; Ec-LF4 & Ec-PA, E. coli cellsharboring pnirB-LF4 mixed with cells containing pnirB-PA63 withoutboost; Ec-LF4 & boost, E. coli cells harboring pnirB-LF4 with a boosterapplication at the same dose a month postimmunization; Ec-LF4 & Ec-PA &boost, E. coli cells harboring pnirB-LF4 mixed with cells containingpnirB-PA63 with a booster application at the same dose a monthpostimmunization; Ec & boost, E. coli cells harboring pnirBVaxin with abooster application a month postimmunization as a negative control;pCLF4 & pCPA & boost, IM injection of pCLF4 and pCPA DNA with a boostone month postimmunization. Open bar, sera harvested a monthpostimmunization before any booster applications; Stippled bar, seraharvested two months postimmunization without any booster applications;solid bar, sera harvested two months postimmunization with one boost amonth prior to bleeding. Shown are geometric means with 8 animals pergroup.

FIG. 9. The lambda P_(R) as a potent promoter for antigen production inE. coli-vectored epicutaneous vaccination.

FIG. 9 is a bar graph showing ELISA titers from: Control, E. coli cellsharboring pλtsP_(R)Vaxin; CMV, E. coli cells harboring pCMV-LF7 andpCPA; nirB, E. coli cells harboring pnirB-LF7 and -PA63; λ32, E. colicells harboring pλtsP_(R)-LF7 and -PA63 with the P_(R) promoterrepressed by cI857 at 32° C.; λ42, E. coli cells harboring pktsP_(R)-LF7and -PA63 with the P_(R) promoter induced for 2 hours at 42° C. prior toadministration. There were 15 mice per group for nirB; and 10 mice pergroup for others. Open bar, sera harvested a month postimmunizationbefore the boost; solid bar, sera harvested two months postimmunization(i.e., a month postboost). Shown are geometric means.

FIG. 10. Neutralizing antibodies against anthrax PA and LF elicited byintranasal and topical application of E. coli vectors expressing PA63and LF7, respectively.

FIGS. 10A and 10B depict the percent cell viability as a function ofserum dilution. Sera was obtained from groups immunized as described inthe figure legends. Lambda, PA63 and LF7 expressed from atemperature-sensitive lambda promoter; NIVS, noninvasive vaccinationonto the skin; IN, intranasal vaccination.

FIG. 11. Protection of mice against inhalation anthrax by topicalapplication of E. coli-vectored epicutaneous vaccines.

FIGS. 11A and 11B depict the percentage of animals surviving afterchallenge with B. anthracis. The animals received topical application ofvaccinations according to the figure legends.

FIG. 12. Induction of apoptosis and death of RAW 264.7 cells bygermaxin.

Cells were exposed to PBS, E. coli control protein or germaxin asindicated. Amount of apoptosis is depicted on the X-axis, and cell deathis shown in the Y-axis.

FIG. 13. Specificity of Germaxin for Induction of Apoptosis.

T-cells in splenocytes and macrophges in lung lavage were marked withanti-mouse CD3 and anti-mouse CD11b, respectively. The cells wereexposed to PBS, E. coli control protein or germaxin as shown prior toimmuocytochemical staining. Arrows indicate apoptosis of macrophages inlung lavage. T cells in splenocytes were not affected.

DETAILED DESCRIPTION

In one embodiment of the present invention, novelgermination/outgrowth-associated proteins are identified. These novelproteins include, but are not limited to: a Bacillus anthracisgermination/outgrowth-associated protein corresponding to Spot 12,wherein the protein has a peptide fingerprint mass spectra as depictedin FIG. 3A, a Bacillus anthracis germination/outgrowth-associatedprotein corresponding to Spot 13, wherein the protein has a peptidefingerprint mass spectra as depicted in FIG. 3B, a Bacillus anthracisgermination/outgrowth-associated protein corresponding to Spot 19,wherein the protein has a peptide fingerprint mass spectra as depictedin FIG. 3C, a Bacillus anthracis germination/outgrowth-associatedprotein corresponding to Spot 15, wherein the protein has a peptidefingerprint mass spectra as depicted in FIG. 3D, a Bacillus anthracisgermination/outgrowth-associated protein corresponding to any of spots21, 24, 27, 41, 47, or 48, each of which is identified in FIG. 2C ashaving increases or decreases by at least 30%; and a spore proteincorresponding to any of spots that has not been reported to be expressedin Bacillus anthracis (Table 1 and FIGS. 2A and 2B). A second embodimentof the invention includes methods of identifying and isolating thepresent novel proteins and additional germination/outgrowth-associatedproteins.

In a still further embodiment of the present invention, the novelgermination/outgrowth-associated and other spore proteins may be used indecontamination of anthrax spores, and the prevention or treatment ofinhalational anthrax, gastrointestinal anthrax, or cutaneous anthrax.Methods and compositions used in the prevention or treatment of diseasearising from exposure to B. anthracis includes the use of vaccinesand/or immunogenic compositions which comprise one or more of the sporeproteins described herein, or fragments thereof, and/or one or morevectors which encode one or more of the spore proteins described herein,or fragments thereof, and/or inhibitors of one or more of thegermination/outgrowth-associated proteins described herein.

Additionally, the invention includes a method of providing immunizationin an animal comprising administering a vector that contains andexpresses a nucleic acid molecule encoding the gene product, in anamount effective to induce the response, wherein the immunization and/orsystemic immune response and/or systemic therapeutic response affordsthe animal protection against challenge with B anthracis. In anadvantageous embodiment, the gene product is one or more of the sporeproteins described herein or a fragment thereof.

A further embodiment of the invention includes a method of inducing asystemic immune response or systemic therapeutic response to a geneproduct, in an animal, comprising administering a vector that containsand expresses a nucleic acid molecule encoding the gene product, in anamount effective to induce the response, wherein the immunization and/orsystemic immune response and/or systemic therapeutic response affordsthe animal protection against challenge with B. anthracis. In anadvantageous embodiment, the gene product is one or more of the sporeproteins described herein or a fragment thereof.

In a still further embodiment, any of the vaccines or immunogeniccompositions herein described may optionally include an excipient and/oradjuvant and/or a suitable carrier or diluent. It is understood that oneof skill in the art would be able to practice the present inventionwithout any undue experimentation.

Yet further still, the present invention provides methods of making theherein described vaccines and immunogenic compositions. Additionally, afurther embodiment of the present invention includes kits providing thecomponents of any of the herein described vaccines or immunogeniccompositions, and methods of making such kits.

The present invention advantageously comprises a method of treatmentafter suspected or true exposure to B. anthracis comprisingadministering an effective amount of one or more inhibitors to one ormore of the spore proteins described herein. Such treatment could alsoinclude administering the aforementioned vaccines or immunogeniccompositions. A further embodiment of the present invention includes thepharmaceuticals, therapeutics, and other compositions employed in thepost-exposure treatment, which can include one or more of the sporeproteins described herein, or fragments thereof, DNA encoding theproteins or fragments thereof, or inhibitors to the fragments or to anyassociated factors produced in conjunction with the spore proteins. Asone of skill in the art is aware, such treatment or therapies mayinclude the use of excipients, diluents, carriers, or other additionalmaterial as is known in the art.

It has been previously demonstrated that vectored vaccines can beinoculated in a novel way as skin-targeted non-invasive vaccines, orimmunological or therapeutic compositions. The combination of vectoredvaccines with a non-invasive delivery mode results in a new class of“democratic” vaccine, or immunological or therapeutic compositions thatrequire little or no special skill and equipment for administration.Thus, one can administer such compositions to the skin of himself orherself (and, this administration can advantageously be under thedirection of a medical practitioner, e.g., to ensure that dosage isproper) or to the skin of an animal (e.g., advantageously a shaved areaof skin if the animal is a mammal, although as demonstrated herein, hairremoval is not necessary, and more advantageously at a region where theanimal will not remove the administration by rubbing, grooming or otheractivity); and, the present invention thus provides advantages in theadministration of vaccine, or immunological, or therapeutic compositionscomprising a vector that expresses a gene product, especially withrespect to administering such compositions to newborns, young animals,animals generally, children and the like, to whom invasive, e.g.,needle, administration can be difficult or inconvenient or painful orharmful.

The present invention is also directed to a method of non-invasiveimmunization or treatment in an animal, comprising the step of:contacting skin of the animal with a recombinant vector in an amounteffective to induce immune response in the animal. Specifically, thepresent invention is directed towards methods and compositions for thetreatment or prevention of infection by Bacillus anthracis.

As used herein, a vector is a tool that allows or facilitates thetransfer of an entity from one environment to another. By way ofexample, some vectors used in recombinant DNA techniques allow entities,such as a segment of DNA (such as a heterologous DNA segment, such as aheterologous cDNA segment) and/or heterologous protein, to betransferred into a target cell. In an advantageous embodiment, thevector includes a viral vector, a bacterial vector, a protozoan vector,a DNA vector, or a recombinant thereof.

In a still further embodiment of the present invention, the therapies,treatments, vaccines, and immunogenic compositions herein described mayall be used to prevent disease due to exposure to B. anthracis inanimals. In an advantageous embodiment of the present invention, theanimal is a vertebrate. In a still more advantageous embodiment of thepresent invention, the vertebrate is a mammal. In an even moreadvantageous embodiment of the present invention, the mammal is a humanor a companion or domesticated or food- or feed-producing or livestockor game or racing or sport animal.

Information in U.S. Pat. No. 5,990,091 issued Nov. 23, 1999, WO99/60164, WO98/00166, “Adenoviral gene delivery elicits distinctpulmonary-associated T helper cell responses to the vector and to itstransgene” (van Ginkel 1997), “Vaccination against acute respiratoryvirus infections and measles in man” (Osterhaus and de Vries 1992), WO99/53940 and U.S. Pat. Nos. 6,042,838 and 6,004,802, can be relied uponfor the practice of this invention (e.g., expressed products, antibodiesand uses thereof, vectors for in vivo and in vitro expression ofexogenous nucleic acid molecules, exogenous nucleic acid moleculesencoding epitopes of interest or antigens or therapeutics and the like,promoters, compositions comprising such vectors or nucleic acidmolecules or expressed products or antibodies, dosages, inter alia). Itis noted that immunological products and/or antibodies and/or expressedproducts obtained in accordance with this invention can be expressed invitro and used in a manner in which such immunological and/or expressedproducts and/or antibodies are typically used, and that cells thatexpress such immunological and/or expressed products and/or antibodiescan be employed in in vitro and ex vivo applications, e.g., such usesand applications can include diagnostics, assays, ex vivo therapy (e.g.,wherein cells that express the gene product and/or immunologicalresponse are expanded in vitro and reintroduced into the host oranimal), etc., see U.S. Pat. No. 5,990,091, WO 99/60164, WO 98/00166, WO99/53940, and U.S. Pat. Nos. 6,042,838, and 6,004,802, and documentscited therein and documents cited or referenced in such documents.Further, expressed antibodies or gene products that are isolated fromherein methods, or that are isolated from cells expanded in vitrofollowing herein administration methods, can be administered incompositions, akin to the administration of subunit epitopes or antigensor therapeutics or antibodies to induce immunity, stimulate atherapeutic response and/or stimulate passive immunity. The quantity tobe administered will vary for the patient (host) and condition beingtreated and will vary from one or a few to a few hundred or thousandmicrograms, e.g., 1 μg to 1 mg, from about 100 ng/kg of body weight to100 mg/kg of body weight per day and preferably will be from 10 pg/kg to10 mg/kg per day. A vector can be non-invasively administered to apatient or host in an amount to achieve the amounts stated for geneproduct (e.g., epitope, antigen, therapeutic, and/or antibody)compositions. Of course, the invention envisages dosages below and abovethose exemplified herein, and for any composition to be administered toan animal or human, including the components thereof, and for anyparticular method of administration, it is preferred to determinetherefor: toxicity, such as by determining the 50% lethal dose (LD₅₀) ina suitable animal model e.g., rodent such as mouse; and, the dosage ofthe composition(s), concentration of components therein and timing ofadministering the composition(s), which elicit a suitable response, suchas by titrations of sera and analysis thereof, e.g., by ELISA and/orseroneutralization analysis. Such determinations do not require undueexperimentation from the knowledge of the skilled artisan, thisdisclosure and the documents cited herein. And, the invention alsocomprehends sequential administration of inventive compositions orsequential performance of herein methods, e.g., periodic administrationof inventive compositions such as in the course of therapy or treatmentfor a condition and/or booster administration of immunologicalcompositions and/or in prime-boost regimens; and, the time and mannerfor sequential administrations can be ascertained without undueexperimentation. Further, the invention comprehends compositions andmethods for making and using vectors, including non-replicative vectors,including methods for producing gene products and/or immunologicalproducts and/or antibodies in vivo and/or in vitro and/or ex vivo (e.g.,the latter two being, for instance, after isolation of cells from a hostthat has had a non-invasive administration according to the invention,e.g., after optional expansion of such cells), and uses for such genesand/or immunological products and/or antibodies, including indiagnostics, assays, therapies, treatments, and the like. Vectorcompositions are formulated by admixing the vector with a suitablecarrier or diluent; and, gene product and/or immunological productand/or antibody compositions are likewise formulated by admixing thegene and/or immunological product and/or antibody with a suitablecarrier or diluent; see, e.g., U.S. Pat. No. 5,990,091, WO 99/60164, WO98/00166, WO 99/53940, and U.S. Pat. Nos. 6,042,838 and 6,004,802,documents cited therein, and other documents cited herein, and otherteachings herein (for instance, with respect to carriers, diluents andthe like).

If nasal or respiratory (mucosal) administration is desired,compositions may be in a form and dispensed by a squeeze spraydispenser, pump dispenser or aerosol dispenser. Such dispensers may alsobe employed to deliver the composition to oral or oral cavity (e.g.,buccal or perlingual) mucosa. Aerosols are usually under pressure bymeans of a hydrocarbon. Pump dispensers can preferably dispense ametered dose or, a dose having a particular particle size.

Compositions of the invention can contain pharmaceutically acceptableflavors and/or colors for rendering them more appealing, especially ifthey are administered orally (or buccally or perlingually); and, suchcompositions can be in the form of tablets or capsules that dissolve inthe mouth or which are bitten to release a liquid for absorptionbuccally or perlingually (akin to oral, perlingual or buccal medicamentsfor angina such as nitroglycerin or nifedimen). The viscous compositionsmay be in the form of gels, lotions, ointments, creams and the like(e.g., for topical and/or mucosal and/or nasal and/or oral and/or oralcavity and/or perlingual and/or buccal administration), and willtypically contain a sufficient amount of a thickening agent so that theviscosity is from about 2500 to 6500 cps, although more viscouscompositions, even up to 10,000 cps may be employed. Viscouscompositions have a viscosity preferably of 2500 to 5000 cps, sinceabove that range they become more difficult to administer. However,above that range, the compositions can approach solid or gelatin formswhich are then easily administered as a swallowed pill for oralingestion and/or a pill or capsule or tablet for holding in the mouth,e.g., for buccal or perlingual administration.

Liquid preparations are normally easier to prepare than gels, otherviscous compositions, and solid compositions. Additionally, liquidcompositions are somewhat more convenient to administer, especially byinjection or orally or buccally or perlinually, to animals, children,particularly small children, and others who may have difficultyswallowing a pill, tablet, capsule or the like, or in multi-dosesituations. Viscous compositions, on the other hand, can be formulatedwithin the appropriate viscosity range to provide longer contact periodswith mucosa, such as the lining of the stomach or nasal mucosa or forperlingual or buccal or oral cavity absorption.

Obviously, the choice of suitable carriers and other additives willdepend on the exact route of administration and the nature of theparticular dosage form, e.g., liquid dosage form (e.g., whether thecomposition is to be formulated into a solution, a suspension, gel oranother liquid form), or solid dosage form (e.g., whether thecomposition is to be formulated into a pill, tablet, capsule, caplet,time release form or liquid-filled form).

Solutions, suspensions and gels, normally contain a major amount ofwater (preferably purified water) in addition to the antigen,lipoprotein and optional adjuvant. Minor amounts of other ingredientssuch as pH adjusters (e.g., a base such as NaOH), emulsifiers ordispersing agents, buffering agents, preservatives, wetting agents,jelling agents, (e.g., methylcellulose), colors and/or flavors may alsobe present. The compositions can be isotonic, i.e., it can have the sameosmotic pressure as blood and lacrimal fluid.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions may be maintained at the selected levelusing a pharmaceutically acceptable thickening agent. Methylcellulose ispreferred because it is readily and economically available and is easyto work with. Other suitable thickening agents include, for example,xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer,and the like. The preferred concentration of the thickener will dependupon the agent selected. The important point is to use an amount whichwill achieve the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf-life of the compositions. Benzyl alcohol may be suitable,although a variety of preservatives including, for example, parabens,thimerosal, chlorobutanol, or benzalkonium chloride may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

Those skilled in the art will recognize that the components of thecompositions must be selected to be chemically inert with respect to thevector or antigen or epitope of interest and optional adjuvant or otheractive or immunity-enhancing ingredients. This will present no problemto those skilled in chemical and pharmaceutical principles, or problemscan be readily avoided by reference to standard texts or by simpleexperiments (not involving undue experimentation), from this disclosureand the documents cited herein.

The immunologically effective compositions of this invention areprepared by mixing the ingredients following generally acceptedprocedures. For example the selected components can be simply mixed in ablender, or other standard device to produce a concentrated mixturewhich can then be adjusted to the final concentration and viscosity bythe addition of water or thickening agent and possibly a buffer tocontrol pH or an additional solute to control tonicity. Generally the pHmay be from about 3 to 7.5. Compositions can be administered in dosagesand by techniques well known to those skilled in the medical andveterinary arts taking into consideration such factors as the age, sex,weight, and condition of the particular patient or animal, and thecomposition form used for administration (e.g., solid vs. liquid).Dosages for humans or other mammals can be determined without undueexperimentation by the skilled artisan, from this disclosure, thedocuments cited herein, the Examples below and from the applications,patents and other documents cited herein and documents cited orreferenced in documents cited herein, all of which are incorporatedherein by reference.

Suitable regimes for initial administration and booster doses or forsequential administrations also are variable, and may include an initialadministration followed by subsequent administrations; but nonetheless,may be ascertained by the skilled artisan, from this disclosure, thedocuments cited and incorporated by reference herein, includingapplications and patents cited herein and documents referenced or citedherein, all of which are hereby incorporated herein by reference, aswell as the Examples below. The compositions can be administered alone,or can be co-administered or sequentially administered with othercompositions of the invention or with other prophylactic or therapeuticcompositions.

EXAMPLES

The present invention will now be described by way of the followingexamples, which are illustrative only and non-limiting.

Bacterial Strains and Spore Preparations.

The Sterne strain of Bacillus anthracis harboring the plasmid (pXO1⁺)encoding the anthrax toxins but lacking the plasmid (pXO2⁻) encoding thecapsule were used in this study (provided by Colorado Serum). Sporeswere prepared from broth cultures of Bacillus anthracis Sterne andpurified by centrifugation on density gradients of Renografin-60 (BraccoDiagnostic, Princeton N.J.) as described previously (Steichen 2003).Dormant spores of Bacillus anthracis Sterne were heated at 65° C. for 30min prior to growth or purification. The germination period afterinoculation of dormant spores into medium was determined by decline inoptical density at 560 m (OD₅₆₀), loss of refractility of spores byphase-contrast microscopy, increase in stainability of spores withGram's crystal violet, and loss of heat resistance as described (Welkos2001).

Example 1 Characterization of Germination of Bacillus anthracis Spores

-   -   -   -   -   Dormant spores of Bacillus anthracis Sterne strain                    were heated at 65° C. for 30 min prior to growth in                    medium for 2 days at 37° C. with shaking. Aliquots                    of samples were removed periodically for                    spectrophotometric analysis (OD₅₆₀) and the results                    were plotted as a growth curve characterizing the                    spore germination stage (FIGS. 1A and 1B). The                    decline in OD₅₆₀ for the first 15 min (FIG. 2 b) is                    recognized as germinating stage (Welkos, S. et al.,                    Microbiology 147:1677-1685, 2001).

Microscopic validation of spore germination was also performed. Purifieddormant spores and spores with germination and early outgrowth inducedat 37° C. in medium for 10 min, respectively, were fixed in 10% bufferedformalin, dried, and viewed on a Zeiss Axioskop2 plus microscope using a100×oil immersion lens.

Example 2 Proteomic Profiling of Bacillus anthracis Spores

Proteomic profiling of Bacillus anthracis dormant and germinating sporeswas used to reveal a number of spore proteins.

Bacillus anthracis spores were prepared as previously described(Steichen, C. et al., J Bacteriol 185:1903-1910, 2003). Total proteinwas extracted from dormant and germinating spores as described (Huang2003). Aliquots containing 300 μg protein were mixed 1:1 withrehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% SB3-10, 5 mM tri-butylphosphine, 1.6% pH 5-8 Bio-lytes, 0.4% pH 3-10Bio-Lytes, and trace bromophenol blue. Samples were subjected toisoelectricfocusing (IEF) in 13-cm linear gradient ImmobilineDry-Strips, pH 4-7, at 60 kVh using a Pharmacia Hoefer Multiphor IIelectrophoresis chamber.

Following IEF, Dry-Strips were incubated at room temperature for 20 minin equilibration solution containing 50 mM Tris-HCl, pH 8.8, 6 M urea,2% sodium dodecyl sulfate (SDS), 30% glycerol, and 5 mMtri-butylphosphine. Dry-Strips were then embedded in 1% agarosecontaining trace bromophenol blue and loaded onto a large format (12.5cm×20 cm), 8-16% gradient SDS-polyacrylamide gel. Electrophoresis wasconducted at 200V for 5 to 6 h or 30 mA per gel overnight until thebromophenol blue dye front was within 2 cm of the bottom of the gel.

Polyacrylamide gels were then stained with Coomassie blue or silvernitrate as described (Huang 2003). Silver-stained gels were scannedusing a Molecular Dynamics Personal Densitometer, and protein spots werequantified using PDQuest software (Bio-Rad, Hercules, Calif.). Alldifferences in gel-spot density between groups were verified manually torule out the possibility of artifacts. To address variability insilver-staining, individual gel spot volumes were normalized by dividingtheir optical density values by the total optical density values of allthe spots present in the gel. FIGS. 2A and 2B depict a proteomic profileobtained by analyzing protein from dormant spores. Fifty protein spotswere subsequently excised and analyzed by in-gel trypsin digestionfollowed by MALDI-TOF MS.

In-gel digestion was performed essentially as described (Huang 2003).Protein spots excised from the Coomassie blue- or silver-stained gelwere destained in 0.2 ml acetonitrile for 15 min and dried to completionin a SpeedVac vacuum centrifuge. Samples were then rehydrated on ice for45 min in digestion buffer (50 mM acetonitrile, 0.04 mg/ml modifiedtrypsin; Promega, Madison, Wis., USA). After removing excess solution,proteins were further digested at 37° C. for 15 h. The resultantpeptides were extracted with 5% formic acid in 50% acetonitrile anddesalted and concentrated using ZipTips containing C 18 resin(Millipore, Bedford, Mass., USA).

Peptides were eluted from the ZipTips with 75% acetonitrile/0.1%trifluoroacetic acid, applied to the sample target, and air-dried.Peptide fragments were then reconstituted in matrix solution containingα-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitrile/0.1%trifluoroacetic acid and analyzed with a PerSeptive Voyager-DE MALDI-TOFmass spectrometer (PerSeptive Biosystems, Framingham, Mass., USA).Peptides were evaporated with a N₂ laser at 337 nm. Each spectrum wasthe cumulative average of 50-100 laser shots. All peptide samples weremeasured as mono-isotopic masses, and autolytic peaks of trypsin wereused for internal calibration. Up to one missed trypsin cleavage wasallowed, although most matches did not contain any missed cleavages.This procedure resulted in mass accuracies of 100 ppm. Peptidefingerprint mass spectra exceeding 5% of full scale were analyzed,interpreted, and matched to SWISS-PROT database entries using Mascot, asearching algorithm available at the Matrix Science, Ltd. Homepage,http://www.matrixscience.com. Matches were computed using aprobability-based Mowse score defined as −10*log (P), where P is theprobability that the observed match was a random event (Perkins 1999).Mowse scores greater than 70 were considered significant (p<0.05).

Peptide mass fingerprint spectra were analyzed, interpreted, and matchedto SWISS-PROT database entries using the Mascot database searchingalgorithm (Table 1). Over 500 proteins from the dormant Sterne sporestrain were reproducibly displayed across an IEF range of 4 to 7. Atotal of 50 protein spots (FIGS. 2A and 2B and Table I), correspondingto 36 different anthrax spore proteins, were successfully identified byMALDI-TOF MS (PerSeptive Biosystems, Framingham, Mass.). Four proteins(spot 11-14) were further sequenced by Q-TOF 2 MS/MS (Micromass,Manchester, UK).

Tandem mass spectral analyses were performed with a Q-TOF 2 massspectrometer (Micromass, Manchester, UK) using electrospray ionization.Samples had undergone a 16 h tryptic digest at 37° C. The resultingpeptides were purified using ZipTips to concentrate and desalt thesamples. The samples were then analyzed by LCMSMS. Liquid chromatographywas performed using a LC Packings Ultimate LC, Switchos microcolumnswitching unit and Famos autosampler (LC Packings, San Francisco,Calif.) The samples were concentrated on a 300 μm i.d. C18 precolumn ata flow rate of 10 μl/min with 0.1% formic acid and then flushed onto a75 μm i.d. C18 column at 200 μl/min with a gradient of 5-100%actonitrile (0.1% formic acid) in 30 min. The nano-lc interface was usedto transfer the lc eluent into the mass spectrometer. The Q-TOF wasoperated in the automatic switching mode whereby multiply-charged ionswere subjected to MSMS if their intensities rose above 6 counts.

Proteins of diverse function were characterized, including thoseinvolved as structural proteins, proteases, amino acid/proteinmetabolism, molecular chaperones, energy metabolism,transcription/translation, transporters, and membrane proteins.

Example 3 Determination of Germination/Outgrowth-Associated Proteins

Germination/outgrowth-associated proteins were revealed by subtractingbackground proteins in dormant spores. The protein samples from dormantspores (time 0, FIG. 2A) and germinating stage (10 min, FIG. 2B) weresubjected to IEF within linear pH gradients ranging from 4-7 followed by2-DE and silver-staining. Using MALDI-TOF MS in conjunction with aprobability-based database searching algorithm, 10 proteins wererecognized to display increase or decreases during the germinatingstage. The protein spots were quantified using PDQuest software(Bio-Rad, Hercules, Calif.). All germination/outgrowth-stage proteinspots showing a statistically significant increase or decrease (definedas greater than 30% change in comparison to those in dormant spores)were selected for further analysis.

To further identify protein spots, spots were cut from thesilver-stained gels and in-gel digested with the trypsin enzyme. Thedigested peptides were mixed with matrix solution containingα-cyano-hydroxycinnamic acid and then analyzed on a PerSeptiveVoyager-DE MALDI-TOF mass spectrometer (PerSeptive Biosystems). Peptidefingerprint mass spectra generated from MALDI-TOF were interpreted andmatched to SWISS-PROT database entries using Mascot, a searchingalgorithm available at the website of Matrix Science, Ltd.,http://www.matrixscience.com.

Overall, a partial proteome map of B. anthracis spores was constructedwith the results generated by 2-DE. Using PDQuest software, 587 pairedprotein spots, isolated from dormant and germinating spores,respectively, were reproducibly displayed under the specified conditions(FIGS. 2A and 2B). Fifty protein spots, comprising 36 individualproteins, were identified by MALDI-TOF MS (Table 1), with proteinsequence coverage averaging 26%. A subset of spots (11-14) was furthercharacterized by Q-TOF 2 MS/MS (Table 1). Comparative sequence alignmentof the 36 identified proteins using the BLAST algorithm (Gish and States1993) showed that 26 of them share the highest sequence identity withputative homologues of B. cereus, 5 are most similar to proteins ofother Bacillus species, whereas 5 have no significant homology todatabase entries. The identified proteins are functionally diverse and86% (31/36) of them have not been included in the B. anthracis proteomicdatabase prior to this report (Table 1). Ninety-four % (29/31) of thesenewly-discovered B. anthracis proteins (Spots 12-14, 17-20, 22-23, 25,29-50) were predicted through whole-genome sequencing efforts (Read2002; Ariel 2003; Ivanova 2003; Read 2003); however, it was not knownthat they are expressed as spore proteins in such abundance.

Twenty-six B. anthracis spore proteins, whose levels are notsignificantly different between dormant and germinating spores, wereidentified as reference markers in this study. Among these, surfacelayer homology (SLH) domain protein is a structural, cellsurface-associated protein (Couture-Tosi. E. 2002) identified byMALDI-TOF MS analysis of protein spots 1-11. Three internal sequences(amino acids, 194-205: AEAAQFLALTDK, 194-206: AEAAQFIALTDKK, and238-249: LSADDVTLEGDK) within spot 11 were sequenced by Q-TOF 2 MS/MS toconfirm the existence of this protein (Table I). Clp protease is astress-responsive protease (Lemos and Burne 2002) identified from spot20. The relevance of chaperones to the germination process is suggestedfrom the number and types identified in this study although some of themmay have been trapped into spores during their expression in mothercells. Of these, we identified Heat shock protein (Hsp)-70 (spot 25),Hsp-60 (spot 26), and chaperonin (spot 28). In addition to chaperones,proteins with a role in transcription and translation were alsoidentified. These include elongation factors G and Ts and RNApolymerase, spots 29, 30, and 31, respectively. Metabolic proteinsaccount for 21 of the 50 identified spots. The enzyme Cysteine synthaseA (EC 4.2.99.8) was identified from protein spots 22 and 23. Acetatekinase (EC 2.7.2.1) was identified from spot 32.Delta-1-pyrroline-5-carboxylate dehydrogenase (EC 1.2.1.3) wasidentified from spots 33 and 34. Multiple subunits of the Pyruvatedehydrogenase multienzyme complex (PDH) were also identified. The PDHE1α and β subunits, the PDH E2 subunit, and the PDH E3 subunit, wereidentified from spots 35, 42, 36, and 38, respectively. A role for theE1β, E2 and E3 subunits of PDH in the regulation of sporulation has beenpreviously elucidated in B. subtilis (Walter and Aronson 1999; Gao2002). Alkyl hydroperoxide reductase (EC 1.6.99.3) was identified fromspot 37. Present in both spores and growing cells, Alkyl hydroperoxidereductase is an oxidative stress-responsive protein and component of theperoxide-responsive perR operon (Casillas-Martinez 2000). Oxidoreductase(EC 1.1.1.-), identified from spot 39, is a member of the largealdo/keto reductase family. Enolase (EC 4.2.1.11) was identified fromspot 40. The F₀ portion of ATP synthase (EC 3.6.3.14) was identifiedfrom spot 45. Spot 43 was characterized as Fructose bisphosphatealdolase (EC 4.1.2.13). In B. subtilis, induction of this enzyme occursfollowing exposure to anaerobic conditions (Marino 2000). Spot 44 wasidentified as Triosephosphate isomerase (EC 5.3.1.1). Spot 50 wasidentified as Alcohol dehydrogenase (EC 1.1.1.1). Band 7, SPFH domainprotein of the Band 7 family, was identified from spot 49. Sporeproteins in this category also include Hypothetical proteins 2 (Spot14), 4 (Spot 17) and 5 (Spot 18) with no significant homology todatabase entries and no known functions.

Seven proteins exhibited germination and early outgrowth-associatedincreases. Of these, the level of Immune inhibitor A (spot 12) wasincreased nearly 2-fold. The GPR-like spore protease (spot 19) wasincreased to a lesser degree. The level of the chaperones Trigger factorin spot 24 and TCP-1/cpn60 in spot 27 was also elevated nearly 2-fold.Trigger factor plays a role in the process of protein maturation and theexport of secretory proteins (Deuerling 1999). Levels of the F₁ portionof ATP synthase (spot 41), Glyceraldehyde-3-phosphate dehydrogenase(spots 46 and 47), and Sugar ABC transporter (spot 48) were allincreased, suggesting that there may be increased metabolic activityinvolving these proteins during spore germination and early outgrowth.

Proteins with germination and early outgrowth-associated decreasesinclude Hypothetical protein 1 which has recently been named germaxin(spot 13), Hypothetical protein 3 (spot 15 and 16), and Alanine racemase(spot 21). The Hypothetical protein 3 was recently identified as apotential B. anthracis spore coat protein (Lai 2003) with unknownfunctions.

Spot 12

Sixteen peaks within the peptide mass fingerprint of spot 12 matchedthose of protein with accession # gi|21399197 in the SWISS-PROT database(FIG. 3A). The protein originally named as hypothetical proteinpredicted by GeneMark is a predicted protein from genomes of Bacillusanthracis (Read 2002). By using a Q-TOF 2 mass spectrometer, theinternal sequence of this predicated protein was determined for theamino acids between positions 450 and 463 with m/z value at 1465.77(FIG. 4B). All 14 amino acid residuals (GIGLATYLDQSVTK) from spot 12were easily determined. More interestingly, the entire amino acidsequence of spot 12 of Bacillus anthracis was found to share more than95% of its identity with immune inhibitor A (gi|9858110) of Bacillusthuringiensis. Both of these proteins contain the HEXXH motif which isdefined as a zinc-binding domain of metalloprotease (Lovgren 1990),suggesting that spot 12 may be a zinc-dependent metalloprotease,specifically immune inhibitor A.

Immune inhibitor A is a secreted virulence protease from Bacillusthuringiensis and it specifically degrades two antibacterial proteins(cecropins and attacins) produced by an insect host (Dalhammar andSteiner 1984), suggesting that it may contribute to the overallvirulence of Bacillus thuringiensis. Bacillus thuringiensis is highlyresistant to the insect immune system due to its production of twofactors, inhibitor A and inhibitor B, which selectively block thehumoral defense system developed by insects against Escherichia coli andBacillus cereus (Edlund 1976). Immune inhibitor A has been known to be ametalloprotease with similarity to the Bacillus thermoproteolyticus'thermolysin, the Pseudomonas aeruginosa elastase and the protease E-15from Serratia (Dalhammar and Steiner 1984). Using a transcriptionalimmune inhibitor A′-lacZ fusion, it was shown that this proteinexpression is activated at the onset of sporulation (Grandvalet 2001).These results have shown for the first time that immune inhibitor A isexpressed in Bacillus anthracis (FIGS. 2, 3, and 4) and that it is ableto be expressed in spores with an increase during germination/outgrowth.

Spot 13

Eight peaks within the peptide mass fingerprint of spot 13 matched thoseof the protein with accession # gi|21399194 in the SWISS-PROT database(FIG. 3B). Like spot 12, spot 13 was named as a hypothetical proteinpredicted by GeneMark and also a predicted protein from the genome ofBacillus anthracis (Read T. D. et al., Science 296:2028-2033, 2002). ByQ-TOF 2 mass spectrometer, we have also sequenced seven internalsequences of this predicated protein (Table 1). One of internal sequencewith 15 amino acid residuals (QSEPVYETTLADLQK) from spot 13 was shown inFIG. 4C. This protein was listed as Hypothetical protein 1 with unknownfunctions (Huang 2004). It has recently been demonstrated that thisprotein induces apoptosis of macrophages (FIG. 13) and the protein hasbeen assigned the name “germaxin”.

Spot 19

Similarly, spot 19 was excised and analysed, and it was determined thatalthough the peptide mass fingerprint of spot 19 matched adihydropyridine sensitive L-type calcium channel beta subunit (accession# gi|21402368) (FIG. 3C) the entire sequence of this protein shares 69%identity to the germination protease, the product of the gpr gene ofBacillus megaterium (Sanchez-Salas and Setlow 1993). This protein hasonly 37% homology with rat dihydropyridine sensitive L-type calciumchannel beta subunit (P54287). The DNA contained by the spores of thebacillus species was saturated with a group of small, acid-solubleproteins (SASP) that protect the DNA from a variety of harsh treatmentsand play a major role in spore resistance and long-term survival. TheSASP-specific germination protease GPR degrades SASP, which alsofunctions as the major energy reserve of the dormant spore, to aminoacids during the first minute of B. subtilis spore germination(Tovar-Rojo 2003). SASP degradation is accompanied by increases intranscription, protein synthesis, and rapid growth (Sanchez-Salas 1992).This degradation was initiated by a sequence-specific protease calledgermination protease, which exhibits no obvious mechanistic or aminoacid sequence similarity to any known class of proteases (Sanchez-Salas1992). The germination protease is synthesized during sporulation as aninactive tetrameric zymogen termed P(46), which later auto-processes toa smaller form termed P(41), which is active only during sporegermination. According to published literature, the crystal structure ofP(46) has been determined (Ponnuraj 2000). Bacillus subtilis mutantswith an inactivated gpr⁻ gene grew, sporulated, and triggered sporegermination as did gpr⁺ strains. However, SASP degradation was very slowduring germination of gpr⁻ mutant spores, and in rich media the timetaken for spores to return to vegetative growth was much longer in gpr⁻than in gpr⁺ spores (Sanchez-Salas and Setlow 1993).

Blocking the function of the GPR-like spore protease might be expectedto produce a phenotype similar to that observed in B. megaterium and B.subtilis gpr mutants, namely decreased SASP degradation withaccompanying retardation of germination (Sanchez-Salas and Setlow 1993).These observations collectively suggest that immunologic orpharmacologic targeting of this enzyme could deter spore germination.Degradation of the cortex, coat, and exosporium during spore germination(Liu 2004) may provide a unique window for drugs or vaccines topenetrate into spores for inactivation of core proteins that arebiologically active.

Although this germination protease has not been documented until now inBacillus anthracis, we found this protein was increased in the 2D Gelduring the germinating stage, suggesting that this protein may play animportant role in anthrax germination.

Spot 15

Five peaks within the peptide mass fingerprint of spot 15 matched thoseof the protein with accession # gi|21399147 in the SWISS-PROT database(FIG. 3D). Spot 15 only has an unknown protein name and may be a noveland anthrax-specific protein since its entire amino acid sequence failsto match proteins of other species.

Spot 21

Alanine racemase (EC 5.1.1.1) (spot 21) converts L-alanine, agermination-promoting compound to D-alanine, a germination inhibitor andessential component of the peptidoglycan found within the cell wall andspore cortex. It is hypothesized that Alanine racemase convertsL-alanine to D-alanine in a hostile environment to inhibit germinationand maximize survival potential (Kanda-Nambu 2000). Previous effortshave demonstrated that Alanine racemase is present in the exosporiumlayer (the outermost layer of spores in contact with the environment) ofB. anthracis (Steichen 2003), and is both tightly adsorbed to exosporiumand a major chemical trigger of germination in B. cereus (Todd 2003).Down-regulation of Alanine racemase during germination as revealed inthis study (Table 1 and FIGS. 2A and 2B) corroborates the hypothesisthat inhibition of this enzyme may be a prerequisite for B. anthracisspores to germinate. Expression of Alanine racemase in exosporium mayallow inactivation of this enzyme by specific antibodies or inhibitorswithout the requirement for penetration into spores, and therebyminimizing survival potential of B. anthracis by triggering pre-maturegermination before the spore reaches a favorable environment.Anti-Alanine racemase agents may even be used for decontamination ofanthrax spores by inducing abortive germination.

Other Isolated Spots

Spot 48 was identified as L-arabinose transport ATP-binding proteinaraG. The araG protein belongs to the ABC transporter and plays a rolein metabolizing L-arabinose as the sole carbon and energy source(Sa-Nogueira and Ramos 1997). Two molecular chaperones (heat shockprotein 60; spot 26 and cpn 60; spot 27) and two energy-involved enzymes(ATP synthase; spot 41 and glyceraldehyde 3-phosphate dehydrogenase(GAPDH), C terminal domain; spot 47) have been up-regulated during sporegermination. The elevated expression of molecular chaperones ingerminating plant seeds suggests that the presence of higher levels ofmolecular chaperones is necessary to assist the rapid assembly of theoligomeric protein structures (Apuya 2001). In addition to the tenproteins that are regulated in expression during B. anthracis sporegermination, we identified forty other proteins that are expressed inboth dormant and germinating spores including a number of novel proteinsdemonstrated for expression in B. anthracis for the first time (Table 1and FIGS. 2A and 2B). This proteomic database provides fundamentalinformation for understanding the biology of anthrax spores. Some ofthese proteins may be essential to the viability of spores, and may alsoappear as novel candidates for drug and vaccine development.

Example 4 Construction of Replication Competent Adenovirus (RCA)-Free AdVectors Encoding B. anthracis PA63 and LF7

Shuttle plasmids pAdApt-PA63 and pAdApt-LF7 containing the B. anthracisPA63 and LF7 fragments, respectively, were constructed by insertion ofPA63 and LF7 fragments into pAdApt (provided by Crucell). PA63 containsa biologically active portion (amino acids 175 to 764) of B. anthracisPA corresponding to the protease-cleaved PA63 fragment of thefull-length 83-KD protein (Price, 2001). LF7 is an immunogenic butatoxic fragment of LF. The catalytic domain of LF resides in theC-terminal part where a HEXXH zinc-metalloprotease consensus sequence(residues 686-690) was identified. Mutations of the critical residuesabolish lethal toxin activity and binding of Zn²⁺ to LF (Mock and Fouet2001). Toxicity of LF was inactivated in LF7 by substitution of Cysteinefor E-687 within the catalytic site of LF (Klimpel 1994). RCA-freeE1/E3-defective adenovirus serotype 5 (Ad5) vectors encoding PA63(rAdCMV-PA63) and LF7 (rAdCMV-LF7) were generated by co-transfectingpAdApt-PA63 and pAdApt-LF7, respectively, with the Ad backbone plasmidpJM17 (Shi 2001) (provided by F. Graham) into PER.C6 packaging cells(Murakami 2002) (provided by Crucell) followed by plaque purifications.RCA-free Ad vectors are generated using this protocol because PER.C6cells contain Ad5 nucleotides 459-3510, which precludes doublecrossover-type homologous recombination with pAdApt-based plasmids thatdo not contain any overlapping sequences (Murakami 2002). Elimination ofRCA in Ad stocks reduces the risk of exposure to the potential oncogeneE1a and pathogenesis induced by replication of Ad in the host.

Example 5 Immunization of Mice Against PA and LF by Intranasal andTopical Application of Ad-Vectored Vaccines

To determine whether animals can be effectively immunized against theanthrax toxin by intranasal and topical application of Ad-vectoredvaccines, AJ and ICR mice were immunized as outlined in Table 2.Briefly, three-month-old mice were immunized by intranasal and topicalapplication of Ad-LF7, Ad-PA63, and a control Ad vector AdCMV-tetC asdescribed (Shi 2001). Vectors, dosages, and frequencies (Groups A-O) aredescribed in C2 and Table 2. Specifically, immunizations comprised oneof: Ad-LF7, an Ad5 vector encoding anthrax LF7; Ad-PA63, an Ad5 vectorencoding anthrax PA63; or AdCMV-tetC, an Ad5 vector encoding the tetanustoxin C-fragment (used here as a control) (Shi 2001). The AJ mice weresusceptible to B. anthracis Sterne spores (Welkos and Friedlander 1988),whereas the ICR mice were more resistant. Animals were immunized on Day0, followed by two booster applications on Days 30 and 210,respectively. TABLE 2 Immunization of mice by Ad-vectored anthraxvaccines Group Strain Immunization A AJ Topical application of 10⁸ pfuof Ad-LF7 B AJ Topical application of 10⁷ pfu of Ad-LF7 C AJ Topicalapplication of 10⁶ pfu of Ad-LF7 D AJ Topical application of 10⁸ pfu ofAdCMV-tetC E AJ Intranasal instillation of 10⁷ pfu of Ad-LF7 F AJTopical application of 10⁸ pfu of Ad-PA63 G AJ Topical application of10⁸ pfu of Ad-LF7 and 10⁸ pfu of Ad-PA63 H AJ Intranasal instillation of10⁷ pfu of Ad-PA63 I ICR Topical application of 10⁸ pfu of Ad-LF7 J ICRTopical application of 10⁷ pfu of Ad-LF7 K ICR Topical application of10⁸ pfu of AdCMV-tetC L ICR Intranasal instillation of 10⁷ pfu of Ad-LF7M ICR Topical application of 10⁸ pfu of Ad-PA63 N ICR Topicalapplication of 10⁸ pfu of Ad-LF7 and 10⁸ pfu of Ad-PA63 O ICR Intranasalinstillation of 10⁷ pfu of Ad-PA63

Example 6 Protection of Mice by Ad-Vectored Vaccines Against B.anthracis Sterne Spores

Control and immunized animals were challenged with B. anthracis Sternespores. Live spores were inoculated into AJ mice by intranasalinstillation, to simulate inhalational anthrax. ICR mice were veryresistant to inhalational Sterne spores, and challenge studies in thismouse strain were thus carried out by intraperitoneal injection (i.p.)of live spores. Three-month-old mice were immunized by intranasal andtopical application of Ad-LF7, Ad-PA63, and a control Ad vectorAdCMV-tetC as described (Shi 2001). Vectors, dosages, and frequencies(Groups A-O) are described in C2 and Table 2. One month after the lastboost when mice were 11 months old, AJ mice were challenged byintranasal instillation of B. anthracis Sterne spores in a volume of 10μl water containing 10⁵ cfu (colony-forming units). ICR mice werechallenged by i.p. injection of Sterne spores in a volume of 50 μl watercontaining 10⁸ cfi. The 50% lethal dose (LD₅₀) of Sterne spores was 10⁴cfu for 3-month-old AJ mice after inhalation; and 10⁷ cfu for3-month-old ICR mice after i.p. injection. This B. anthracis Sterne 34F2vaccine strain (Colorado Serum Company, Denver, Colo.) isnonencapsulated (pXO2⁻) but toxigenic (pXO1⁺) with PA and LF expressedfrom the pXO1 plasmid. Spores were grown in the supplemented nutrientbroth medium (Hanson 1964) with modifications [0.8% Bacto nutrientbroth, 0.1% KCl, 0.012% MgSO₄, 1 mM Ca(NO₃)₂, 10 μM MnCl₂, 1 μM FeSO₄,pH 7.6] at 37° C. with shaking. Dormant anthrax spores were purifiedfrom death-phase (48-h) culture by centrifugation of spores, vegetativecells and cell debris through gradients of Renografin-60 (BraccoDiagnostic, Princeton, N.J.) in conjunction with extensive washes asdescribed (Steichen 2003). Purified dormant anthrax spores were storedin water at 4° C.

Challenged animals were monitored daily for survival up to 30 days.Difficult breathing was taken as the disease endpoint. No symptoms ordeaths occurred 14 days post-challenge. As shown in FIG. 5A-5E, the Advector encoding the immunogenic but atoxic LF fragment (LF7) wassignificantly more effective than its counterpart encoding PA63 inprotecting mice (both AJ and ICR) against live Sterne spores, and theAd-vectored nasal vaccine is more potent than its epicutaneouscounterpart. The data depicted in FIG. 5A-5E was plotted as % deathversus days after challenge. Numbers in parentheses after each Grouprepresent the number of animals that received each treatment.

Intranasal instillation of 10⁷ pfu of Ad-LF7 was able to protect 89% AJ(Group E in FIG. 5A) and 77% ICR (Group L in FIG. 5C) mice against alethal dose of anthrax spores. Topical application of 108 pfu Ad-LF7 wasable to protect 50% AJ (Group A in FIG. 5A) and 36% ICR (Group I in FIG.5C) mice. Intranasal and topical application of Ad-PA63 was not veryeffective in protecting animals (Groups F and H in FIG. 5B; Group M and0 in FIG. 5D). Co-administration of Ad-LF7 and Ad-PA63 (Group G in FIG.5B; Group N in FIG. 5D) was as effective as Ad-LF7 alone. Greater than87% animals immunized with the control vector AdCMV-tetC (Group D inFIG. 5A; Group K in FIG. 5C) were killed by anthrax spores within 9days.

These results demonstrate that an Ad vector encoding an immunogenic butatoxic LF fragment (LF7) is more effective in protecting animals againstinhalational anthrax than a counterpart encoding PA63. Eighty-nine % and50% AJ mice could be protected against inhalational anthrax followingintranasal instillation and topical application of Ad-LF7, respectively.PA63 was less immunogenic than LF7 in the mouse genetic background.

Example 7 Elicitation of Anti-PA and -LF Antibodies by Ad-VectoredAnthrax Vaccines

To determine whether protection against anthrax as shown in FIG. 5A-5Ewas mediated, at least in part, by the elicitation oftoxin-neutralization antibodies following administration of Ad-vectoredanthrax vaccines, anti-PA and -LF antibody titers were analyzed by ELISAand mouse macrophage protection assay through toxin neutralization.

A/J (Groups A-H) and ICR (Groups I-O) mice were immunized against PA andLF as described in Examples 5 and 6, above. Serum samples were harvested1, 2, and 8 months after the primary immunization, and analyzed foranti-PA and -LF antibodies by ELISA as described (Shi 2001) withpurified PA and LF proteins (List Biological Laboratories, Inc.),respectively, as the capture antigen (1 ng protein per well). FIG. 6A-6Dshows that Ad-LF7 elicited higher ELISA antibody titers than Ad-PA63.Additionally, Ad-vectored nasal vaccine was more effective in inducingan antibody response than its epicutaneous counterpart. These resultsshow that the level of ELISA antibody titers was correlated with that ofprotection (FIG. 5A-5E).

Additionally, serum samples harvested 8 months after the primaryimmunization (one month after the last booster application) wereanalyzed for toxin-neutralization antibodies as described for a mousemacrophage protection assay (Price 2001) with modifications. Briefly,sera from all immunized animals in a group were pooled in equal amounts,serially diluted, and incubated with PA and LF proteins (List BiologicalLaboratories, Inc.). A final concentration of 0.06 μg PA and 0.12 μg LFwith or without neutralization was added to 10⁵ mousemonocyte-macrophage RAW 264.7 cells (ATCC) per well in a 96-well plate.At the end of the incubation 16 hour incubation period, 0.05 μg MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma)per well was added, followed by a 1-h incubation. Cells that surviveexposure to PA-LF are able to oxidize MTT to an insoluble purplepigment, thus providing a proportional measure of the viability of thecells. At the end of the incubation period, the level of survival wasdetermined using a microplate reader at OD₅₄₀. Shown in FIG. 7A-7C isthe percent cell viability. Survival of control cells without exposureto PA+LF was arbitrarily defined as 100%. FIG. 7A-7C shows that Ad-LF7elicited higher toxin-neutralization antibody titers than Ad-PA63. Also,the Ad-vectored nasal vaccine was more effective in inducing an antibodyresponse than its epicutaneous counterpart.

Overall, as shown in FIGS. 6A-6D and 7A-7C, the highest ELISA andtoxin-neutralization antibody titers were induced by intranasalinstillation of Ad-LF7. Topical application of Ad-LF7 was also quiteeffective, whereas the immunogenicity of PA63 was low. The level ofELISA antibody titers (FIG. 6A-6D) and toxin-neutralization antibodytiters (FIG. 7A-7C) were both correlated with that of protection (FIG.5A-5E). Due to the close correlation between toxin-neutralizationantibody titers and protection of animals against live anthrax spores,it is thus logical to use the toxin-neutralization antibody titer as astandard in determining the potency of an experimental anthrax vaccine.

Example 8 Dissemination and Degradation of PA DNA Following TopicalApplication of Ad-PA63

To analyze the safety margin of Ad-vectored epicutaneous anthraxvaccines, DNA was extracted from various tissues of AJ mice at differenttime points following topical application of Ad-PA63, followed byPCR-based amplification of the PA DNA. Specifically, AJ Mice wereimmunized by topical application of 108 pfu of Ad-PA63 as described inExamples 5 and 6, above. At indicated time points, total DNA wasextracted from the tissues and amplified by PCR using specific primers.The forward primer 5′-AGTACAAGTGCTGGACCTACGGTTCCAGAC-3′ and reverseprimer 5′-TCCTATCTCATAGCCTTTTTTAGAAAAGAT-3′ were used to amplify thenearly full-length 1.7 kb PA63 fragment. The forward primer5′-TATCCTTCTAAAAACTTGGCGCCAATCGCA-3′ and reverse primer5′-GATCACTAGGATTAACCGCCGCTATCCGCC-3′ were used to amplify a 0.3 kbsubfragment encompassing 18% of the PA63 fragment. The data werepresented as the number of animals containing detectable signals for aspecific tissue per total number of animals analyzed. DNA was extractedusing DNAZOL solution. One μg of DNA was amplified for 39 cycles atoptimized annealing temperatures. Amplified DNA fragments werefractionated in 1% agarose gel and stained with ethidium bromide.

As summarized in Table 3, the nearly full-length PA63 gene could beamplified from the skin at the administration site both 3 hours and 1day post-inoculation. The nearly full-length gene was undetectable inskin DNA after 2 days or in DNA extracted from other tissues. However, asubfragment of the PA63 DNA was amplified from liver, spleen, heart,lung, brain, kidney, whole blood, or lymph nodes using a different setof primers. The PA63 subfragment was amplifiable from a wide variety oftissues 1 day postimmunization, but not 3 hr after topical application.No foreign DNA was detectable in any of the tissues a monthpost-inoculation. Results suggest that vector DNA delivered by a vaccinepatch may be acquired by putative antigen-presenting cells (APC),followed by degradation and dissemination into deep tissues includinglymphoid organs. The elimination of foreign DNA in a month postimmunization highlights the safety of epicutaneous vaccines. Thetransient expression of antigens in the skin following topicalapplication of vectors may also minimize antigen-induced apoptosis of Tlymphocytes that may deplete the pool of memory T cells (Swain 1999).

These results show that the mouse skin can absorb Ad-vectored vaccines.Ad vectors absorbed by the skin is rapidly degraded and disseminatedsystemically. One month postimmunization, no trace amount of the antigenDNA could be amplified by PCR in any of the tissues examined, indicatingthat the skin is able to protect the host's genetic integrity bydegrading absorbed environmental DNA. This leads to the conclusion thattopical application of Ad vectors is safe because the vector DNA doesnot persist. TABLE 3 Summary of PA63 DNA dissemination in AJ micefollowing topical application of Ad-PA63 Skin^(a) LN^(b) Heart SpleenLiver Kidney Blood Brian Lung I. full-length PA63 fragment 3 hr 3/3 0/30/3 0/3 0/3 0/3 0/3 0/3 0/3 1 day 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 1month 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 II. Subfragment of PA63 DNA 3hr 3/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 1 day 3/3 3/3 3/3 2/3 3/3 2/3 3/31/3 2/3 1 month 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3^(a)Administration site (abdominal skin);^(b)pooled inguinal, cervical, and brachial lymph nodes.

Example 9 Construction of Recombinant Plasmids for Expression of B.anthracis Antigens in E. coli Vectors

The plasmid pnirBVaxin with the E. coli nirB promoter inserted upstreamfrom a multiple cloning site (MCS) was constructed by amplifying thenirB promoter including its ATG initiation codon and ribosome bindingsite with polymerase chain reaction (PCR) from plasmid pTET-nir(Chatfield 1992) (provided by J. McGhee). The amplified fragmentcontaining the nirB promoter was then inserted into the AflIII-XhoI siteof plasmid pZErO™-2 (Invitrogen) to generate plasmid pZErO-nirB.Amplification of a MCS from plasmid pBluescript II KS (Stratagene) withPCR was also performed, and the amplified MCS was inserted into theXhoI-Stul site of pZErO-nirB to generate plasmid pZErO-nirB-MCS. PlasmidpnirBVaxin was generated by the insertion of a synthetic T7 terminatorinto the SacI-StuI site of pZErO-nirB-MCS to generate. The T7 terminatorwas made by annealing the following two synthetic oligonucleotides:CCATAACCC CTTGGGGCCT CTAAACGGGT CTTGAGGGGT TTTTTGCTGA AAGGAGG and TCGAGGTATTGGG GAACCCCGGAGATTTGCCCA GAACTCCCCA AAAAACGACT TTCCTCC.

The plasmid pλtsP_(R)Vaxin was constructed by replacing the nirBpromoter in plasmid pnirBVaxin with a fragment containing thebacteriophage lambda P_(R) promoter-cro ribosome binding site-ATG codonin conjunction with the λcI857 variant of the XcI gene which codes for atemperature-sensitive repressor of P_(R). The c1857 product repressesP_(R) at 32° C., but allows transcription from the P_(R) promoter at 42°C. (Queen 1983). The lambda PR promoter-cI857 repressor unit wasamplified from plasmid pCQV2 (Queen 1983) (provided by C. Queen) andinserted into the AflIII-XhoI site of pnirBVaxin to replace the nirBpromoter.

Recombinant plasmids encoding the B. anthracis PA63 were constructed byamplifying the PA63 gene with PCR from plasmid pCPA (provided by D.Galloway) which contains a biologically active portion (corresponding toamino acids 175 to 764) of B. anthracis PA corresponding to theprotease-cleaved PA63 fragment of the full-length 83-KD protein (Price2001). The PA63 fragment was inserted into the XhoI-XbaI site ofpnirBVaxin and pλtsP_(R)Vaxin to generate plasmids pnirB-PA63 (PA63driven by nirB promoter) and pλtsP_(R)-PA63 (PA63 driven by λP_(R)promoter), respectively.

Recombinant plasmids encoding the N-terminal region (amino acids 10-254containing the PA binding domain) of B. anthracis LF (LF4 fragment) wereconstructed by excising the LF4 fragment from plasmid pCLF4 (Price 2001)(provided by D. Galloway) followed by insertion into the XhoI-NotI siteof pnirBVaxin and pλtsP_(R)Vaxin to generate plasmids pnirB-LF4 (LF4driven by nirB promoter) and pλtSP_(R)-LF4 (LF4 driven by λP_(R)promoter), respectively. The LF4 fragment was amplified by PCR fromplasmid pCLF4 and inserted into the BamHI-XhoI site of pCAL-n-FLAG togenerate plasmid pCAL-n-FLAG-LF4 (LF4 driven by the T7 promoter).

Recombinant plasmids encoding the full length but atoxic B. anthracis LF(LF7 fragment) were constructed by amplifying the LF7 fragment with PCRfrom plasmid pAdApt-LF7 (provided by D. Galloway) followed by insertionof the LF7 fragment into the BamHI-XhoI site of pCAL-n-FLAG to generateplasmid pCAL-n-FLAG-LF7 (LF7 driven by T7 promoter). The LF7 fragmentwas subsequently excised from pCAL-n-FLAG-LF7 and inserted into theBamHI-SacI site of pnirBVaxin and pλtsP_(R)Vaxin to generate plasmidspnirB-LF7 (LF7 driven by nirB promoter) and pλtsP_(R)-LF7 (LF7 driven bykPR promoter), respectively. The LF4 fragment in pCLF4 was replaced bythe LF7 fragment to generate plasmid pCMV-LF7 (LF7 driven by thecytomegalovirus [CMV] early promoter). The catalytic domain of LFresides in the C-terminal part where a HEXXH zinc-metalloproteaseconsensus sequence (residues 686-690) is identified. Mutations of thecritical residues abolish lethal toxin activity and binding of Zn²⁺ toLF (Mock, 2001). Toxicity of LF was inactivated in LF7 by substitutionof Cysteine for E-687 within the catalytic site of LF (Klimpel 1994).

Example 10 Immunization of Animals by Topical Application of E.coli-Vectored Vaccines

To determine whether animals can be immunized by topical application ofE. coli-vectored vaccines and whether vectors expressing PA and LF alsoconfer synergy when combined, as previously demonstrated for DNA-basedvaccines (Price 2001), mice were immunized by topical application of E.coli vectors expressing LF4, PA63, or both, and determination ofantibody titers against LF was subsequently performed (FIG. 8).

Young (2-3 months old) female ICR mice (Harlan) were immunized bytopical application of E. coli vectors harboring the plasmids pnirB-LF4,pnirB-PA63, or a combination of both. Topical application was carriedout by pipetting 5×10⁹ cfu of E. coli BL21-CodonPlus cells (Stratagene)harboring each plasmid onto pre-shaved abdominal skin of a mousefollowed by covering the vector as a thin film over naked skin with apiece of the Tegaderm patch (3M) as described (Shi 2001). E. coli cellswere harvested during mid-log phase and washed twice in PBS prior toadministration. Unabsorbed vectors were washed away in an hour. As apositive control, DNA-based vaccination was conducted by intramuscular(IM) injection of pCLF4 and pCPA DNA (Price 2001) (100 μg each) into thehind leg quadriceps as described (Shi 2001). Serum samples were assayedfor anti-LF antibodies 1 and 2 months after the primary immunization.Titers of anti-LF IgG were determined by ELISA as described (Shi 2001)using purified LF protein (provided by D. Galloway) as the captureantigen. Briefly, serum samples and peroxidase-conjugated goatanti-mouse IgG (Promega) were incubated sequentially on the plates withextensive washing between each incubation. The end-point was calculatedas the dilution of serum producing the same OD₄₉₀ as a 1/100 dilution ofpreimmune serum. Sera negative at the lowest dilution tested wereassigned endpoint titers of 1. FIG. 8 shows the tier results obtainedone month post immunization but prior to any boosts, two months postimmunization but prior to any boosts, and two month post immunizationwhere a boost was give one month post immunization.

This demonstrates that animals can be immunized against an anthraxantigen following topical application of a recombinant E. coli vectorexpressing the antigen and suggests that the cutaneous immune system maybe rapidly activated when the outer layer of skin is in contact with E.coli cells at the specified concentration. Further, an unidentified hostdefense mechanism may subsequently disrupt E. coli cells, capture theantigen from within, and present the exogenous antigen to the hostimmune system in eliciting an immune response. The commercial impact ofthis finding is great owing to the potency of this regimen (as this ismore effective than IM injection of DNA), simplicity in mass productionof E. coli vectors, elimination of problems associated with needleinjections, and the high biosafety margin of laboratory E. coli strains.

In an attempt to optimize the potency of E. coli-vectored epicutaneousvaccines, the anthrax antigen was inserted downstream from a variety ofprokaryotic and eukaryotic promoters as described above, followed byepicutaneous immunization of animals with E. coli vectors harboring thesame antigen driven by different promoters, and subsequently determinedthe anti-LF antibody response (see FIGS. 9 and 10A-10B).

A/J mice were immunized by topical application of recombinant E.coli-vectored vaccines with one booster application a monthpostimmunization, as described in Example 6. Specifically, 5×10⁹ cfu ofE. coli BL21-CodonPlus cells harboring a plasmid encoding LF7 mixed withthe same number of E. coli cells harboring another plasmid encoding PA63driven by the same promoter was administered onto the skin of eachanimal. Serum samples were assayed for anti-LF antibodies 1 and 2 monthsafter the primary immunization. Animals in the control group receivedadministrations of E. coli cells harboring pλtsP_(R)Vaxin.

These results are shown in FIG. 9 which shows that thetemperature-induced lambda PR promoter is potent in eliciting an immuneresponse against the expressed antigen, even more effective than the E.coli nirB promoter. LF7 driven by the eukaryotic CMV promoter did notelicit any detectable antibody response. The same pattern of antibodyresponse was reproduced in ICR mice (data not shown). Similar responsesagainst PA were also generated when sera were analyzed with ELISA usingPA83 protein (provided by D. Galloway) as the capture antigen.

It is possible that the potency of E. coli-vectored epicutaneous vaccineis correlated with the level of antigen expression. Thetemperature-induced lambda P_(R) promoter may be more transcriptionallyactive than other promoters tested. Failure of the eukaryotic CMVpromoter to generate an effective epicutaneous vaccine in the E. colisetting suggests that the antigen protein may be expressed in E. colicells instead of the animal's own cells. The temperature-sensitivelambda P_(R) promoter-cI857 repressor provides a unique system forproduction of exogenous antigens that may impede the growth of E. colicells.

Additionally, AJ mice were immunized by intranasal and topicalapplication of E. coli vectors expressing PA63 and LF7. Sera wereserially diluted and incubated with PA+LF, and added to RAW264.7 cellsas described in Example 7. Cell viability was determined by the MTTassay. The data is plotted in FIGS. 10A and 10B, which demonstrates thatthe E. coli-vectored nasal vaccine is more potent than the E.coli-vectored epicutaneous vaccine. Additionally, it is apparent thatLF7 is more immunogenic than PA63.

Furthermore, AJ mice immunized by topical application of E. coli vectorsexpressing LA63 and LF7 driven by the temperature-sensitive lambdapromoter were challenged by intranasal inoculation of B. anthracisSterne spores as described in Example 6, above. The challenge resultsshown in FIGS. 11A and 11B show that topical application of E.coli-vectored epicutaneous vaccines protects animals againstinhalational anthrax.

Example 11 Induction of Apoptosis and Death of RAW 264.7 Cells byGermaxin

The mouse macrophage-like RAW 264.7 cells were incubated for one daywith PBS, 25 ng/ml E. coli control protein or germaxin at concentrationsof 25 ng/ml or 250 ng/ml. Germaxin was obtained by purification throughan affinity column from E. coli cells expressing B. anthracis germaxin.

After incubation, cells were stained with Annexin V to detect apoptosis,and 7-AAD for determining cell death by flow cytometry. As shown in FIG.12, there was significant increase in both apoptosis and cell death inthose cells incubated with either concentration of germaxin incomparison with those incubated with PBS or the E. coli control protein.Additionally, the levels of apoptosis and cell death show littlevariation between cells incubated with the two concentrations ofgermaxin.

Example 12 Specificity of Germaxin to Induce Apoptosis in Macrophages

To determine whether germaxin's ability to induce cell death andapoptosis was specific to any types of cells, lung lavage andsplenocytes were harvested and cultured from an AJ mouse. Macrophagespresent in the lung lavage culture were marked with an antibody againstmouse CD11b (red cells in upper half of FIG. 13), and T-cells present inthe splenocyte culture were marked with an antibody against mouse CD3(red cells in lower half of FIG. 13). Cells were exposed to PBS, 25ng/ml E. coli control protein, or 25 ng/ml B. anthracis germaxin(germaxin is described in Table 1).

One day later, apoptotic cells were identified by the Tunel assay (seearrows in upper right panel of FIG. 13). These results demonstrate thatthe B. anthracis germaxin protein induces the apoptosis of macrophagesbut not of T-cells.

The invention will now be further described by the following numberedparagraphs:

-   -   1. A Bacillus anthracis germination/outgrowth-associated spore        protein corresponding to Spot 12 (Accession # NP_(—)843763),        wherein the protein has a peptide fingerprint mass spectra as        depicted in FIG. 3A.    -   2. A Bacillus anthracis germination/outgrowth-associated spore        protein corresponding to Spot 13 (Accession #: NP_(—)655179),        wherein the protein has a peptide fingerprint mass spectra as        depicted in FIG. 3B.    -   3. A Bacillus anthracis germination/outgrowth-associated spore        protein corresponding to Spot 19 (Accession # NP_(—)846769),        wherein the protein has a peptide fingerprint mass spectra as        depicted in FIG. 3C.    -   4. A Bacillus anthracis germination/outgrowth-associated spore        protein corresponding to Spot 15 (Accession # NP_(—)655132),        wherein the protein has a peptide fingerprint mass spectra as        depicted in FIG. 3D.    -   5. A Bacillus anthracis germination/outgrowth-associated spore        protein that is up- or down-regulated during germination by at        least 30%, wherein the protein corresponds to Spot 21 (Accession        # NP_(—)654182), 24 (Accession # NP_(—)846918), 27 (Accession #        NP_(—)654198), 41 (Accession # NP_(—)847705), 47 (Accession #        NP_(—)653587), or 48 (Accession # NP_(—)846171).    -   6. Novel Bacillus anthracis spore proteins corresponding to        Spots 17 (Accession # NP_(—)657537), 18 (Accession #        NP_(—)658120), 20 (Accession # NP_(—)653598), 28 (Accession #        NP_(—)242212), 49 (Accession # NP_(—)654492), 50 (Accession #        NP_(—)654728).    -   7. A method of immunization in an animal and/or a method of        inducing a systemic immune response or systemic therapeutic        response to a gene product, in an animal, comprising        administering a vector that contains and expresses a nucleic        acid molecule encoding the gene product, in an amount effective        to induce the response, wherein the immunization and/or systemic        immune response and/or systemic therapeutic response affords the        animal protection against challenge with B. anthracis.    -   8. A method of immunization in an animal and/or a method of        inducing a systemic immune response or systemic therapeutic        response to a gene product, in an animal, comprising        administering a vector that contains and expresses a nucleic        acid molecule encoding the gene product, in an amount effective        to induce the response, wherein the nucleic acid molecule        encodes the Bacillus anthracis spore protein of any of        paragraphs 1-6 or a portion thereof alone or in combination.    -   9. A vaccine or immunogenic composition against Bacillus        anthracis comprising a spore protein as described in any of        paragraphs 1-6, or a fragment thereof, or a genetic vector        encoding a spore protein as described in any of paragraphs 1-6,        or a fragment thereof, and optionally an excipient and/or        adjuvant and/or a suitable carrier or diluent.    -   10. A method of preparing the vaccine or immunogenic composition        of paragraph 9.    -   11. A method of treating an animal after suspected or real        exposure to B. anthracis comprising administering an effective        amount of one or more inhibitors to one or more of the spore        proteins of paragraphs 1-6.    -   12. The method of any of paragraphs 7, 8 or 11, wherein the        animal is a vertebrate.    -   13. The method of paragraph 12 wherein the vertebrate is a        mammal.    -   14. The method of paragraph 13 wherein the mammal is a human or        a companion or domesticated or food- or feed-producing or        livestock or game or racing or sport animal.    -   15. A method of immunization, therapy, or decontamination of        anthrax spores by inducing pre-mature spore germination with        agents targeting the spore proteins of paragraphs 1-6.    -   16. The Bacillus anthracis spore protein of any of paragraphs        1-7 wherein the protein is obtained by a process comprising the        steps of expressing the protein in a vector and purifying the        protein.    -   17. The spore protein of paragraph 1, wherein the protein        comprises the sequences depicted in FIG. 3A.    -   18. The spore protein of paragraph 2, wherein the protein        comprises the sequences depicted in FIG. 3B.    -   19. The spore protein of paragraph 3, wherein the protein        comprises the sequences depicted in FIG. 3C.    -   20. The spore protein of paragraph 1, wherein the protein        comprises the sequences depicted in FIG. 3D.    -   21. A method of immunization in an animal and/or a method of        inducing a systemic immune response or systemic therapeutic        response to any of the 31 B. anthracis spore proteins described        in Table 1.    -   22. A method of non-invasive immunization in an animal and/or a        method of inducing a systemic immune response or systemic        therapeutic response to a gene product from Bacillus anthracis,        in an animal, comprising contacting skin, or nasal, oral,        perlingual or buccal mucosa of the animal with a vector that        contains and expresses a nucleic acid molecule encoding the gene        product, in an amount effective to induce the response.    -   23. The method of paragraph 22 wherein the method comprises        contacting the skin of the animal with the vector.    -   24. The method of paragraph 22 wherein the method comprises        contacting the nasal mucosa with the vector.    -   25. The method of paragraph 22 wherein the method comprises        contacting the oral or buccal or perlingual mucosa with the        vector.    -   26. The method of paragraph 22 wherein the nucleic acid molecule        is exogenous or heterologous to the vector.    -   27. The method of paragraph 22 wherein the response comprises a        systemic immune response.    -   28. The method of paragraph 22 wherein the vector comprises and        expresses an exogenous nucleic acid molecule encoding an epitope        of interest.    -   29. The method of paragraph 22 wherein the vector comprises and        expresses an antigen.    -   30. The method of paragraph 22 wherein the vector comprises and        expresses a therapeutic product.    -   31. The method of paragraph 22 wherein the nucleic acid molecule        encodes an epitope of interest and/or an antigen of interest        and/or a nucleic acid molecule that stimulates and/or modulates        an immunological response and/or stimulates and/or modulates        expression comprising transcription and/or translation an        endogenous and/or exogenous nucleic acid molecule.    -   32. The method of paragraph 26 wherein the exogenous nucleic        acid molecule encodes one or more of an antigen or portion        thereof, or one or more of an epitope of interest, from Bacillus        anthracis.    -   33. The method of paragraph 26 wherein the exogenous nucleic        acid molecule encodes one or more of: anthrax protective        antigen, anthrax lethal factor, and anthrax edema factor.    -   34. The method of paragraph 31 wherein the response is induced        by the vector expressing the nucleic acid molecule in the        animal's cells.    -   35. The method of paragraph 34 wherein the cells comprise        epidermal cells.    -   36. The method of paragraph 31 wherein the response comprises an        immune response against Bacillus anthracis.    -   37. The method of paragraphs 22 wherein the animal is a        vertebrate.    -   38. The method of paragraph 37 wherein the vertebrate is a        mammal.    -   39. The method of paragraph 38 wherein the mammal is a human or        a companion or domesticated or food- or feed-producing or        livestock or game or racing or sport animal.    -   40. The method of paragraph 39 wherein the animal is a cow, a        horse, a dog, a cat, a goat, a sheep, or a pig.    -   41. The method of paragraph 22 wherein the vector comprises one        or more of a viral vector, a viral coat comprising a virus        having some or all viral genes deleted therefrom, a bacterial        vector, protozoan vector, a transposon, a retrotransposon, and a        DNA vector.    -   42. The method of paragraph 41 wherein the vector comprises a        recombinant vector.    -   43. The method of paragraph 42 wherein the vector comprises an        adenovirus.    -   44. The method of paragraph 43 wherein the adenovirus is        defective or deleted in its E1 and/or E3 and/or E4 region(s).    -   45. The method of paragraph 41, wherein the vector is an        Escherichia bacterial vector.    -   46. The method of paragraph 45 wherein the Escherichia vector is        Escherichia coli.    -   47. The method of paragraph 46 wherein the Escherichia coli is        replication defective.    -   48. The method of paragraph 47 wherein the Escherichia coli is        rendered replication defective by irradiation, antibiotics,        fixatives, genetic substitutions or deletions, or gentle heat.    -   49. A method of inducing a systemic immune response or systemic        therapeutic response to a gene product from Bacillus anthracis,        in an animal, comprising topical and/or intranasal and/or        mucosal and/or buccal and/or perlingual and/or oral        administration of a vector comprising an adenovirus defective in        its E1 and/or E3 and/or E4 region(s), wherein the adenovirus        contains and expresses a nucleic acid molecule encoding the gene        product for the response.    -   50. A method of inducing a systemic immune response or systemic        therapeutic response to a gene product from Bacillus anthracis,        in an animal, comprising topical and/or intranasal and/or        mucosal and/or buccal and/or perlingual and/or oral        administration of a vector comprising replication defective        Escherichia coli, wherein the vector contains and expresses a        nucleic acid molecule encoding the gene product for the        response.    -   51. The method of paragraph 49 or 50 comprising topical        administration.    -   52. The method of paragraph 49 or 50 comprising intranasal        administration.    -   53. The method of paragraph 49 wherein the adenovirus is        defective or deleted in its E1 and E3 regions.    -   54. The method of paragraphs 49 or 50 wherein the vector        comprises an exogenous or heterologous nucleic acid molecule        encoding the gene product for the response.    -   55. The method of paragraph 49 or 50 wherein the nucleic acid        molecule is exogenous or heterologous and encodes an epitope of        interest and the method is for inducing a systemic immunological        response.    -   56. The method of paragraph 49 wherein the adenovirus is a human        adenovirus.    -   57. The method of paragraph 49 wherein the adenovirus comprises        a non-human adenovirus.    -   58. The method of paragraph 22, 49, or 50 wherein the vector is        matched to, or a natural pathogen of, the animal.    -   59. The method of paragraph 22, 49, or 50 comprising applying a        delivery device including the vector to the skin of the animal.    -   60. The method of paragraph 59 further comprising disposing the        vector in and/or on the delivery device.    -   61. A pharmaceutical, vaccine, immunogenic, immunological or        therapeutic composition for use in a method according to any one        of paragraphs 22, 49, or 50 comprising the vector in a        pharmaceutically acceptable carrier or diluent for administering        the vector topically or mucosally or intranasally or buccally or        perlingually or orally.    -   62. A kit for preparing a pharmaceutical, vaccine, immunogenic,        immunological or therapeutic composition for use in a method        according to any one of paragraphs 22, 49, or 50 comprising in a        first container the vector and in a second container a        pharmaceutically acceptable carrier or diluent for administering        the vector topically or mucosally or intranasally or bucally or        perlingually or orally; wherein the containers are optionally        present in the same package or are in separate packages; and,        the kit optionally contains instructions for admixture of the        vector and the carrier or diluent and/or administration.    -   63. The kit of paragraph 62 further comprising a delivery device        in a third container; wherein the third container is optionally        present in the same package as one or both of the first and        second containers or is in a package separate from the first and        second containers; and, wherein kit optionally contains        instructions for installing the vector or vector and carrier or        diluent into or onto the delivery device and/or for        administration or application of the delivery device.

64. The method of paragraph 23, wherein contacting the skin is performedin combination with the use of microneedles. All publications mentionedin the above specification are here incorporated by reference. Variousmodifications and variations of the described methods and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in immunology or related fields are intended to be withinthe scope of the following claims. TABLE 1 List of proteins identifiedin B. anthracis spores Spot Protein Accession No.^(a) Function Note 1-11SLH, S-layer homology domain NP_654830 Structural *1262.57 (238-249)protein *1277.65 (194-205) *1405.77 (194-206) 65% sequence identity toS-layer protein precursor of B. licheniformis (JC4930) 12 Immuneinhibitor A metalloprotease^(+↑) NP_843763 Protease 94% sequenceidentity to Immune inhibitor A precursor of B. cereus *1465.77 (450-463)13 Hypothetical protein 1^(+↓) NP_655179 Unknown Hypothetical proteinpredicted by GeneMark ™ with no homology to database entries *958.49(58-66) *1008.46 (110-116) *1009.42 (95-104) *1351.55 (92-104) *1721.79(117-131) *1827.90 (134-149) *2044.94 (132-463) 14 Hypothetical protein2⁺ NP_655177 Unknown Hypothetical protein predicted by GeneMark ™ withno homology to database entries *1182.65 (112-121) *1210.63 (86-95) 15,16 Hypothetical protein 3^(↓) NP_655132 Unknown Hypothetical proteinpredicted by GeneMark ™ with no homology to database entries 17Hypothetical protein 4⁺ NP_657537 Unknown Hypothetical protein predictedby GeneMark ™ with no homology to database entries 18 Hypotheticalprotein 5⁺ NP_658120 Unknown Hypothetical protein predicted byGeneMark ™ with no homology to database entries 19 GPR-like sporeprotease^(+↑) NP_846769 Protease 91% sequence identity to Spore proteaseof B. cereus; 67% identity to SASP degradation spore proteinase GPRprecursor of B. subtilis (gi|98324) 20 Clp protease⁺ NP_653598 ProteaseEC 3.4.21.92^(b); 99% sequence identity to Clp P1 of B. thuringiensis 21Alanine racemase^(↓) NP_842805 Metabolism EC 5.1.1.1; 96% sequenceidentity to Alanine racemase of B. cereus (NP_830132) 22, 23 Cysteinesynthase A⁺ NP_842636 Metabolism EC 4.2.99.8; 89% sequence identity toCysteine synthase of B. cereus (NP_829970) 24 Trigger factor^(↑)NP_846918 Molecular EC 5.2.1.8; 90% sequence identity to Trigger factorof B. Chaperone cereus 25 Heat shock protein 70⁺ NP_658346 Molecular 89%sequence identity to Hsp70 (DnaK) of B. cereus chaperone 26 Heat shockprotein 60⁺ BAB68361 Molecular 85% sequence identity to Hsp60 of B.thuringiensis chaperone 27 TCP-1/cpn60 chaperonin family^(↑) NP_654198Molecular 95% sequence identity to 60 kDa chaperonin GRoEL of B.chaperone cereus 28 Class I heat shock protein (chaperonin)⁺ NP_242212Molecular 73% sequence identity to Class I heat shock protein chaperone(chaperonin) of B. subtilis (CAB14489) 29 Elongation factor G,C-terminus⁺ NP_654055 Translation 97% sequence identity to proteintranslation elongation factor G (EF-G) of B. cereus 30 Elongation factorTs⁺ NP_657794 Translation 100% sequence identity to protein translationelongation factor Ts (EF-Ts) of B. cereus (NP_833545) 31 RNA polymerase,alpha chain, N- terminal NP_654079 Transcription EC 2.7.7.6; 96%sequence identity to DNA-directed RNA domain⁺ polymerase alpha chain ofB. cereus (NP_830037) 32 Acetate kinase⁺ NP_658669 Metabolism EC2.7.2.1; 92% sequence identity to acetate kinase of B. cereus(NP_834343) 33, 34 Delta-1-pyrroline-5-carboxylate NP_654240 MetabolismEC 1.2.1.3; 99% sequence identity to Delta-1-pyrroline-5- dehydrogenase,putative/Aldehyde carboxylate dehydrogenase of B. cereus (NP_830183)dehydrogenase family⁺ 35 Pyruvate dehydrogenase complex E1 NP_846421Metabolism EC 1.2.4.1; 85% sequence identity to Pyruvate component,alpha subunit⁺ dehydrogenase complex E1 component, alpha subunit of B.cereus (NP_833692) 36 Pyruvate dehydrogenase complex E2 NP_846419Metabolism EC 2.3.1.-; 82% sequence identity to Dihydrolipoamidecomponent, dihydrolipoamide acetyltransferase component of pyruvatedehydrogenase acetyltransferase⁺ complex of B. cereus (NP_833690) 37Alkyl hydroperoxide reductase, F subunit⁺ NP_842891 Metabolism EC1.6.99.3; 92% sequence identity to Alkyl hydroperoxide reductase, Fsubunit of B. cereus (NP_830215) 38 Pyruvate dehydrogenase complex E3NP_846418 Metabolism EC 1.8.1.4; 94% sequence identity toDihyrolipoamide component, dihydrolipoamide dehydrogenase of B. cereus(NP_833689) dehydrogenase⁺ 39 Oxidoreductase, aldo/keto reductaseNP_845729 Metabolism EC 1.1.1.-; 92% sequence identity to2,5-diketo-D-gluconic family⁺ acid reductase of B. cereus (NP_833127) 40Enolase⁺ NP_653583 Metabolism EC 4.2.1.11; 98% sequence identity toEnolase of B. cereus (NP_834403) 41 ATP synthase F1 beta subunit^(+↑)NP_847705 Metabolism EC 3.6.3.14; 95% sequence identity to ATP synthasebeta chain of B. cereus (NP_834968) 42 Pyruvate dehydrogenase complex E1NP_846420 Metabolism EC 2.2.1.1; 95% sequence identity to Pyruvatecomponent, beta subunit⁺ dehydrogenase E1 component, beta subunit of B.cereus (NP_833691) 43 Fructose-bisphosphate aldolase class-II⁺ NP_653796Metabolism EC 4.1.2.13; 95% sequence identity to Fructose- bisphosphatealdolase of B. cereus (NP_834997) 44 Triosephosphate isomerase⁺NP_653585 Metabolism EC 5.3.1.1; 74% sequence identity toTriosephosphate isomerase of B. megaterium (JQ1955) 45 ATP synthase B/B′CF(0) ⁺ NP_653766 Metabolism EC 3.6.3.14; 80% sequence identity to ATPsynthase beta chain of B. cereus (NP_834972) 46, 47 Glyceraldehyde3-phosphate NP_653587 Metabolism EC 1.2.1.12; 94% sequence identity toGlyceraldehyde 3- dehydrogenase, C-terminal domain^(+↑) phosphatedehydrogenase of B. cereus (NP_834805) 48 Sugar ABC transporter,ATP-binding NP_846171 Metabolism 92% sequence identity to nucleosidetransport ATP-binding protein^(+↑) protein of B. cereus (NP_833512) 49Band 7, SPFH domain/Band 7 family ⁺ NP_654492 Unknown 76% sequenceidentity to Flotillin of B. cereus (NP_830375) 50 Alcohol dehydrogenase,zinc-binding NP_654728 Metabolism EC 1.1.1.1; 86% sequence identity toAlcohol dehydrogenase⁺ dehydrogenase of B. cereus (NP_830592)Ninety-six % (48/50) of protein spots were linked to the published B.anthracis genomesequence(Read 2002; Read 2003); spot 26 was revealed as the B. anthracisHSP60 and spot 28 asthe B. halodurans Class I HSP at the National Center for BiotechnologyInformation (NCBI)database (http://www.ncbi.nlm.nih.gov).^(a)Accession No. are accessible at the NCBI database^(b)(EC -.-.-.-) represents the enzyme commission number*Observed m/z (predicted peptide sequence) using Q-TOF 2⁺Protein expression not previously reported in B. anthracis^(↑)Germination-associated increases^(↓)Germination-associated decreases

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1. A Bacillus anthracis germination/outgrowth-associated spore proteincorresponding to: (i) Spot 12 (Accession # NP_(—)843763), wherein theprotein has a peptide fingerprint mass spectra as depicted in FIG. 3A;(ii) Spot 13 (Accession #: NP_(—)655179), wherein the protein has apeptide fingerprint mass spectra as depicted in FIG. 3B; (iii) Spot 19(Accession # NP_(—)846769), wherein the protein has a peptidefingerprint mass spectra as depicted in FIG. 3C; or (iv) Spot 15(Accession # NP_(—)655132), wherein the protein has a peptidefingerprint mass spectra as depicted in FIG. 3D.
 2. A Bacillus anthracisgermination/outgrowth-associated spore protein that is up- ordown-regulated during germination by at least 30%, wherein the proteincorresponds to Spot 21 (Accession # NP_(—)654182), 24 (Accession #NP_(—)846918), 27 (Accession # NP_(—)654198), 41 (Accession #NP_(—)847705), 47 (Accession # NP_(—)653587), or 48 (Accession #NP_(—)846171).
 3. Novel Bacillus anthracis spore proteins correspondingto Spots 17 (Accession # NP_(—)657537), 18 (Accession # NP_(—)658120),20 (Accession # NP_(—)653598), 28 (Accession # NP_(—)242212), 49(Accession # NP_(—)654492), 50 (Accession # NP_(—)654728).
 4. A methodof immunization in an animal and/or a method of inducing a systemicimmune response or systemic therapeutic response to a gene product, inan animal, comprising administering a vector that contains and expressesa nucleic acid molecule encoding the gene product, in an amounteffective to induce the response, wherein the immunization and/orsystemic immune response and/or systemic therapeutic response affordsthe animal protection against challenge with B. anthracis.
 5. A methodof immunization in an animal and/or a method of inducing a systemicimmune response or systemic therapeutic response to a gene product, inan animal, comprising administering a vector that contains and expressesa nucleic acid molecule encoding the gene product, in an amounteffective to induce the response, wherein the nucleic acid moleculeencodes the Bacillus anthracis spore protein of any of claims 1-3 or aportion thereof alone or in combination.
 6. A vaccine or immunogeniccomposition against Bacillus anthracis comprising a spore protein asdescribed in any of claims 1-3, or a fragment thereof, or a geneticvector encoding a spore protein as described in any of claims 1-3, or afragment thereof, and optionally an excipient and/or adjuvant and/or asuitable carrier or diluent.
 7. A method of preparing the vaccine orimmunogenic composition of claim
 6. 8. A method of treating an animalafter suspected or real exposure to B. anthracis comprisingadministering an effective amount of one or more inhibitors to one ormore of the spore proteins of claims 1-3.
 9. The method of any of claims4, 5 or 8, wherein the animal is a vertebrate.
 10. The method of claim 9wherein the vertebrate is a mammal.
 11. The method of claim 10 whereinthe mammal is a human or a companion or domesticated or food- orfeed-producing or livestock or game or racing or sport animal.
 12. Amethod of immunization, therapy, or decontamination of anthrax spores byinducing pre-mature spore germination with agents targeting the sporeproteins of claims 1-3.
 13. The Bacillus anthracis spore protein of anyof claims 1-3 wherein the protein is obtained by a process comprisingthe steps of expressing the protein in a vector and purifying theprotein.
 14. The spore protein of claim 1, wherein the protein comprisesthe sequences depicted in FIG. 3A, FIG. 3B, FIG. 3C or FIG. 3D.
 15. Amethod of immunization in an animal and/or a method of inducing asystemic immune response or systemic therapeutic response to any of the31 B. anthracis spore proteins described in Table
 1. 16. A method ofnon-invasive immunization in an animal and/or a method of inducing asystemic immune response or systemic therapeutic response to a geneproduct from Bacillus anthracis, in an animal, comprising contactingskin, or nasal, oral, perlingual or buccal mucosa of the animal with avector that contains and expresses a nucleic acid molecule encoding thegene product, in an amount effective to induce the response.
 17. Themethod of claim 16 wherein the method comprises contacting the skin ofthe animal with the vector.
 18. The method of claim 16 wherein themethod comprises contacting the nasal mucosa with the vector.
 19. Themethod of claim 16 wherein the method comprises contacting the oral orbuccal or perlingual mucosa with the vector.
 20. The method of claim 16wherein the nucleic acid molecule is exogenous or heterologous to thevector.
 21. The method of claim 16 wherein the response comprises asystemic immune response.
 22. The method of claim 16 wherein the vectorcomprises and expresses an exogenous nucleic acid molecule encoding anepitope of interest.
 23. The method of claim 16 wherein the vectorcomprises and expresses an antigen.
 24. The method of claim 16 whereinthe vector comprises and expresses a therapeutic product.
 25. The methodof claim 16 wherein the nucleic acid molecule encodes an epitope ofinterest and/or an antigen of interest and/or a nucleic acid moleculethat stimulates and/or modulates an immunological response and/orstimulates and/or modulates expression comprising transcription and/ortranslation an endogenous and/or exogenous nucleic acid molecule. 26.The method of claim 20 wherein the exogenous nucleic acid moleculeencodes one or more of an antigen or portion thereof, or one or more ofan epitope of interest, from Bacillus anthracis.
 27. The method of claim20 wherein the exogenous nucleic acid molecule encodes one or more of:anthrax protective antigen, anthrax lethal factor, and anthrax edemafactor.
 28. The method of claim 25 wherein the response is induced bythe vector expressing the nucleic acid molecule in the animal's cells.29. The method of claim 28 wherein the cells comprise epidermal cells.30. The method of claim 25 wherein the response comprises an immuneresponse against Bacillus anthracis.
 31. The method of claims 16 whereinthe animal is a vertebrate.
 32. The method of claim 31 wherein thevertebrate is a mammal.
 33. The method of claim 32 wherein the mammal isa human or a companion or domesticated or food- or feed-producing orlivestock or game or racing or sport animal.
 34. The method of claim 33wherein the animal is a cow, a horse, a dog, a cat, a goat, a sheep, ora pig.
 35. The method of claim 16 wherein the vector comprises one ormore of a viral vector, a viral coat comprising a virus having some orall viral genes deleted therefrom, a bacterial vector, protozoan vector,a transposon, a retrotransposon, and a DNA vector.
 36. The method ofclaim 35 wherein the vector comprises a recombinant vector.
 37. Themethod of claim 36 wherein the vector comprises an adenovirus.
 38. Themethod of claim 37 wherein the adenovirus is defective or deleted in itsE1 and/or E3 and/or E4 region(s).
 39. The method of claim 35, whereinthe vector is an Escherichia bacterial vector.
 40. The method of claim39 wherein the Escherichia vector is Escherichia coli.
 41. The method ofclaim 40 wherein the Escherichia coli is replication defective.
 42. Themethod of claim 41 wherein the Escherichia coli is rendered replicationdefective by irradiation, antibiotics, fixatives, genetic substitutionsor deletions, or gentle heat.
 43. A method of inducing a systemic immuneresponse or systemic therapeutic response to a gene product fromBacillus anthracis, in an animal, comprising topical and/or intranasaland/or mucosal and/or buccal and/or perlingual and/or oraladministration of a vector comprising: (i) an adenovirus defective inits E1 and/or E3 and/or E4 region(s), wherein the adenovirus containsand expresses a nucleic acid molecule encoding the gene product for theresponse; or, (ii) replication defective Escherichia coli, wherein thevector contains and expresses a nucleic acid molecule encoding the geneproduct for the response.
 44. The method of claim 43 comprising topicaladministration.
 45. The method of claim 43 comprising intranasaladministration.
 46. The method of claim 43 wherein the adenovirus isdefective or deleted in its E1 and E3 regions.
 47. The method of claims43 wherein the vector comprises an exogenous or heterologous nucleicacid molecule encoding the gene product for the response.
 48. The methodof claim 43 wherein the nucleic acid molecule is exogenous orheterologous and encodes an epitope of interest and the method is forinducing a systemic immunological response.
 49. The method of claim 43wherein the adenovirus is a human adenovirus.
 50. The method of claim 43wherein the adenovirus comprises a non-human adenovirus.
 51. The methodof claim 16 or 43 wherein the vector is matched to, or a naturalpathogen of, the animal.
 52. The method of claim 16 or 43 comprisingapplying a delivery device including the vector to the skin of theanimal.
 53. The method of claim 52 further comprising disposing thevector in and/or on the delivery device.
 54. A pharmaceutical, vaccine,immunogenic, immunological or therapeutic composition for use in amethod according to any one of claims 16 or 43 comprising the vector ina pharmaceutically acceptable carrier or diluent for administering thevector topically or mucosally or intranasally or buccally orperlingually or orally.
 55. A kit for preparing a pharmaceutical,vaccine, immunogenic, immunological or therapeutic composition for usein a method according to any one of claims 16 or 43 comprising in afirst container the vector and in a second container a pharmaceuticallyacceptable carrier or diluent for administering the vector topically ormucosally or intranasally or bucally or perlingually or orally; whereinthe containers are optionally present in the same package or are inseparate packages; and, the kit optionally contains instructions foradmixture of the vector and the carrier or diluent and/oradministration.
 56. The kit of claim 55 further comprising a deliverydevice in a third container; wherein the third container is optionallypresent in the same package as one or both of the first and secondcontainers or is in a package separate from the first and secondcontainers; and, wherein kit optionally contains instructions forinstalling the vector or vector and carrier or diluent into or onto thedelivery device and/or for administration or application of the deliverydevice.
 57. The method of claim 16 or 43, wherein application of thevector to the skin is performed in combination with the use ofmicroneedles.